PROKARYOTIC RNAi-LIKE SYSTEM AND METHODS OF USE

ABSTRACT

Provided herein are methods for inactivating a target polynucleotide. The methods use a psiRNA having a 5′ region and a 3′ region. The 5′ region includes, but is not limited to, 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. The 3′ region is substantially complementary to a portion of the target polynucleotide. The methods may be practiced in a prokaryotic microbe or in vitro. Also provided are polypeptides that have endonuclease activity in the presence of a psiRNA and a target polynucleotide, and methods for using the polypeptides.

GOVERNMENT FUNDING

The present invention was made with government support under Grant No. R01GM054682, awarded by the NIH. The Government has certain rights in this invention.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser. No. 61/083,616, filed Jul. 25, 2008, Ser. No. 61/180,656, filed May 22, 2009, and Ser. No. 61/227,554, filed Jul. 22, 2009, each of which is incorporated by reference herein.

BACKGROUND

Small, non-coding (nc)RNAs are found in all domains of life and function in a wide array of essential cellular processes. In eukaryotes, small ncRNAs including siRNAs and microRNAs have been shown to function in post-transcriptional gene silencing by targeting exogenous or endogenous RNAs, respectively, in a process called RNA interference, or RNAi (Hannon, 2002, Nature, 418:244-251). Another class of small RNAs referred to as piRNAs (piwi-associated) or rasiRNAs (repeat associated small interfering) regulate spreading of selfish genetic elements such as transposons or repeat elements in organisms including mammals, plants and flies (Kim V. N., 2006, Genes Dev, 20:1993-1997; Nishida and Siomi, 2006, Tanpakushitsu Kakusan Koso, 51:2450-2455; Aravin et al., 2007, Science 318:761-764; Hartig et al., 2007, Genes Dev, 21:1707-1713; Lin H., 2007, Science, 316:397).

An RNAi-like system that functions in genome defense has recently been proposed to exist in prokaryotes (Markova et al., 2006, Biol Direct, 1:7; Deveau et al., 2008, J Bacteriol, 190:1390-1400; Sorek et al., 2008, Nat Rev Microbiol, 6:181-186; Tyson and Banfield, 2008, Environ Microbiol, 10:200-207). The hallmark of the proposed prokaryotic RNAi (or pRNAi) system is the CRISPR locus, a cluster of short direct repeats that separate short variable sequences (i.e. clustered regularly interspaced short palindromic repeat). A number of the variable sequences (also sometimes called “spacers”) found in CRISPR loci display complementarity (or identity) to known prokaryotic viruses, plasmids and transposons (Bolotin et al., 2005, Microbiology, 151:2551-2561; Mojica et al., 2005, Mol Evol, 60:174-182; Pourcel et al., 2005, Microbiology, 151:653-663; Lillestol et al., 2006, Archaea, 2:59-72; Markova et al., 2006, Biol Direct, 1:7). The other signature component of the hypothesized pRNAi system is a set of protein-coding genes referred to as CRISPR-associated or Cas genes that are found in CRISPR-containing genomes (Jansen et al., 2002, Mol Microbiol, 43:1565-1575; Markarova et al., 2002, Nucleic Acids Res, 30:482-496; Haft et al., 2005, PLoS Comput Biol, 1:e60; Markova et al., 2006, Biol Direct, 1:7). The Cas genes are predicted to encode nucleases, helicases, RNA-binding proteins and a polymerase (Jansen et al., 2002, Mol Microbiol, 43:1565-1575; Markarova et al., 2002, Nucleic Acids Res, 30:482-496; Haft et al., 2005, PLoS Comput Biol, 1:e60; Markova et al., 2006, Biol Direct, 1:7). These bioinformatically-predicted properties of the CRISPR and Cas gene products led to the hypothesis that they comprise an RNAi-like system of genome defense in prokaryotes, in which RNAs derived from the variable regions of CRISPR loci (prokaryotic silencing or psiRNAs) guide the silencing (e.g., degradation) of genome invaders by Cas proteins (Bolotin et al., 2005, Microbiology, 151:2551-2561; Lillestol et al., 2006, Archaea, 2:59-72; Markova et al., 2006, Biol Direct, 1:7). The Cas proteins are also expected to function in the processing of the psiRNAs and in the integration of new psiRNA genes (directed against newly encountered pathogens) into the genome.

Recent studies have provided strong evidence for a role of CRISPR loci in viral resistance in prokaryotes. Several groups have observed that virus exposure leads to the appearance of new virus-derived sequence elements within the CRISPR loci of surviving (resistant) isolates (Barrangou et al., 2007, Science 315:1709-1712; Deveau et al., 2008, J Bacteriol, 190:1390-1400; Horvath et al., 2008, J Bacteriol, 190:1401-1412). In addition, Barrangou et al. showed that an alteration of an organism's CRISPR sequences that generates or destroys correspondence with a viral sequence results in viral resistance and viral sensitivity, respectively (Barrangou et al., 2007, Science 315:1709-1712). However, the pathway by which CRISPR loci confer viral resistance remains hypothetical and undefined.

CRISPR loci are present in about half of bacterial genomes and nearly all archaeal genomes (Godde and Bickerton, 2006, J Mol Evol, 62:718-729; Markova et al., 2006, Biol Direct, 1:7). A given locus can contain as few as 2, and as many as several hundred repeat-psiRNA units (Grissa et al., 2007, Bioinformatics, 8:172; Sorek et al., 2008, Nat Rev Microbiol, 6:181-186). The repeat sequences are generally 25 to 45 nucleotides long and often weakly palindromic at the 5′ and 3′ termini (Jansen et al., 2002, Mol Microbiol, 43:1565-1575). Interspersed between the repeats are the variable, putative psiRNA-encoding sequences, which are usually similar in length to the repeats. RNAs arising from CRISPR loci have been detected by RNA cloning and/or Northern blotting in 3 archaeal species: Archaeoglobus fulgidus, Sulfolobus solfataricus and Sulfolobus acidocaldarius (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72). These studies provided convincing evidence of transcription of entire CRISPR loci from the predicted transcriptional leader sequences that are found at one end of the loci, and of a discrete series of smaller RNAs that correspond in length to multiples of repeat-psiRNA units (e.g. ˜70, 140, 210, 280 nts, etc. (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481)). These findings along with RNA sequence analysis led to a hypothesized biogenesis pathway in which primary CRISPR transcripts are endonucleolytically cleaved within repeat sequences to produce psiRNAs flanked by repeat sequence at both the 5′ and 3′ ends (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481).

SUMMARY OF THE INVENTION

Provided herein are methods for inactivating a target polynucleotide in a microbe. The methods may include introducing into the microbe a psiRNA that includes a 5′ region and a 3′ region. The 5′ region may be a psiRNA-tag that includes 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. As described herein, the skilled person can easily identify nucleotides sequences suitable for use as a psiRNA-tag. Optionally, the psiRNA-tag nucleotide sequence is chosen from a CRISPR locus present in the microbe into which the psiRNA is introduced. An example of a useful psiRNA-tag is 5′-AUUGAAAS, wherein S is G or C. The 3′ region may include at least 18 nucleotides, and in some aspects, no greater than 75 nucleotides. The 3′ region is typically substantially complementary, optionally complementary, to a portion of the target polynucleotide.

The target polynucleotide may be cleaved in the region that is substantially complementary to the 3′ region. The psiRNA may be introduced into the cell as an RNA polynucleotide, or as a DNA polynucleotide encoding the psiRNA. In some aspects the psiRNA-tag is 8 nucleotides. The target polynucleotide may be DNA or RNA, and may be an endogenous polynucleotide. Methods described herein may be used, for example, for immunizing a microbe against an exogenous polynucleotide. The target polynucleotide that is inactivated may be the exogenous polynucleotide, or the target polynucleotide may be encoded by the exogenous polynucleotide. In one aspect, the exogenous polynucleotide is a bacteriophage polynucleotide. Also provided herein are genetically modified microbes that include the psiRNAs described herein.

Provided herein are methods for inactivating a target polynucleotide. The methods include incubating under suitable conditions a composition that includes a target polynucleotide, and a psiRNA that includes a 5′ region and a 3′ region. The 5′ region may be a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. An example of a useful psiRNA-tag is 5′-AUUGAAAS, wherein S is G or C. The 3′ region may include 5′-NNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNN (SEQ ID NO:24) or 5′-NNNNNNNNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNNNNNNN (SEQ ID NO:25), wherein the 3′ region is substantially complementary, optionally complementary, to a portion of the target polynucleotide. The composition also contains a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, and a Cmr6 polypeptide. Optionally, the composition may further include a Cmr5 polypeptide. The target polynucleotide may be cleaved in the region that is substantially complementary to the 3′ region of SEQ ID NO:24 or SEQ ID NO:25. In some aspects the psiRNA-tag is 8 nucleotides. The target polynucleotide may be cleaved opposite the position defined by the arrow. The method may be performed in vitro or in vivo. Examples of suitable cells include Pyrococcus furiosus, a Sulfolobus solfataricus, or a S. tokodaii cell.

The amino acid sequence of the Cmr1 polypeptide and the amino acid sequence of SEQ ID NO:4 may have at least 80% identity. The amino acid sequence of the Cmr2 polypeptide and the amino acid sequence of SEQ ID NO:8 may have at least 80% identity. The amino acid sequence of the Cmr3 polypeptide and the amino acid sequence of SEQ ID NO:10 may have at least 80% identity. The amino acid sequence of the Cmr4 polypeptide and the amino acid sequence of SEQ ID NO:12 may have at least 80% identity. The amino acid sequence of the Cmr6 polypeptide and the amino acid sequence of SEQ ID NO:16 may have at least 80% identity. The polypeptides have endonuclease activity, for instance, endoribonuclease activity. The amino acid sequence of an optional Cmr5 polypeptide and the amino acid sequence of SEQ ID NO:14 may have at least 80% identity.

Also provided herein are methods for inactivating a target polynucleotide in a microbe. The methods may include introducing into a microbe a psiRNA that includes a 5′ region and a 3′ region. The 5′ region may be a psiRNA-tag that includes 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. Optionally, the psiRNA-tag nucleotide sequence is chosen from a CRISPR locus present in the microbe into which the psiRNA is introduced. An example of useful psiRNA-tag is 5′-AUUGAAAS, wherein S is G or C. The 3′ region may include at least 18 nucleotides, and in some aspects, no greater than 75 nucleotides. The 3′ region is typically substantially complementary, optionally complementary, to a portion of the target polynucleotide. The psiRNA introduced into the cell as an RNA polynucleotide, or as a DNA polynucleotide encoding the psiRNA. Examples of suitable cells include Pyrococcus furiosus, Sulfolobus solfataricus, or a S. tokodaii cell.

The microbe into which the psiRNA is introduced may be a genetically modified microbe, wherein the genetically modified microbe includes an exogenous Cmr1 polypeptide, an exogenous Cmr2 polypeptide, an exogenous Cmr3 polypeptide, an exogenous Cmr4 polypeptide, and an exogenous Cmr6 polypeptide. The amino acid sequence of the Cmr1 polypeptide and the amino acid sequence of SEQ ID NO:4 may have at least 80% identity. The amino acid sequence of the Cmr2 polypeptide and the amino acid sequence of SEQ ID NO:8 may have at least 80% identity. The amino acid sequence of the Cmr3 polypeptide and the amino acid sequence of SEQ ID NO:10 may have at least 80% identity. The amino acid sequence of the Cmr4 polypeptide and the amino acid sequence of SEQ ID NO:12 may have at least 80% identity. The amino acid sequence of the Cmr6 polypeptide and the amino acid sequence of SEQ ID NO:16 may have at least 80% identity. The polypeptides have endonuclease activity, for instance, endoribonuclease activity. Optionally, the genetically modified microbe may further include a Cmr5 polypeptide. The amino acid sequence of Cmr5 polypeptide and the amino acid sequence of SEQ ID NO:14 may have at least 80% identity.

Further provided herein are compositions that include enriched polypeptides, wherein the polypeptides are a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, a Cmr5 polypeptide, a Cmr6 polypeptide, or a combination thereof. Optionally, the polypeptides are isolated.

Provided herein are enriched, optionally isolated polynucleotides that include a 5′ region and a 3′ region. The 5′ region may be a psiRNA-tag that includes 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer, and the 3′ region may include at least 18 nucleotides, or the complement of the polynucleotides. The polynucleotides may be RNA or DNA. A polynucleotide may further include an additional polynucleotide, for instance, a target polynucleotide, that is hybridized to the 3′ region. Also provided are vectors that include the isolated polynucleotide, and vectors that encode the polynucleotide. Further provided are genetically modified microbes that include such vectors, and genetically modified microbes encoding an exogenous polynucleotide that includes a 5′ region and a 3′ region.

Also provided herein are (a) Cmr1 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively; (b) Cmr1 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:6 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively; (c) Cmr2 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively; (d) Cmr3 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:10 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16, respectively; (e) Cmr4 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:12 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:16, respectively; (f) Cmr5 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:14 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, Cmr 4, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:16, respectively; and (g) Cmr6 polypeptides, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:16 have at least 80% identity, wherein the polypeptide has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, Cmr4, and Cmr5 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14, respectively. Also provided are polynucleotides encoding such polypeptides. Optionally, such polynucleotides also include a heterologous polynucleotide, such as a regulatory sequence operably linked to the polynucleotide, or a vector. A genetically modified microbe, such as E. coli, may include an exogenous polynucleotide encoding one or more of these polypeptides.

Further provided are kits. An example of a kit contains enriched, optionally isolated polypeptides, wherein the polypeptides are a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, a Cmr6 polypeptide, and optimally, a Cmr5 polypeptide.

As used herein, an “enriched” polynucleotide means that a polynucleotide constitutes a significantly higher fraction of the total DNA or RNA present in a mixture of interest than in cells from which the sequence was taken. A person skilled in the art could enrich a polynucleotide by preferentially reducing the amount of other polynucleotides present, or preferentially increasing the amount of the specific polynucleotide, or both. However, polynucleotide enrichment does not imply that there is no other DNA or RNA present, the term only indicates that the relative amount of the sequence of interest has been significantly increased. The term “significantly” qualifies “increased” to indicate that the level of increase is useful to the person using the polynucleotide, and generally means an increase relative to other nucleic acids of at least 2 fold, or more preferably at least 5 to 10 fold or more. The teen also does not imply that there is no polynucleotide from other sources. Other polynucleotides may, for example, include DNA from a bacterial genome, or a cloning vector.

As used herein, an “enriched” polypeptide defines a specific amino acid sequence constituting a significantly higher fraction of the total of amino acids present in a mixture of interest than in cells from which the polypeptide was separated. A person skilled in the art can preferentially reduce the amount of other amino acid sequences present, or preferentially increase the amount of specific amino acid sequences of interest, or both. However, the teen “enriched” does not imply that there are no other amino acid sequences present. Enriched simply means the relative amount of the sequence of interest has been significantly increased. The term “significant” indicates that the level of increase is useful to the person making such an increase. The term also means an increase relative to other amino acids of at least 2 fold, or more preferably at least 5 to 10 fold, or even more. The term also does not imply that there are no amino acid sequences from other sources. Other amino acid sequences may, for example, include amino acid sequences from a host organism.

As used herein, an “isolated” substance, such as a polynucleotide or a polypeptide, is one that has been removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized. For instance, a polypeptide or a polynucleotide can be isolated. A substance may be purified, i.e., is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which it is naturally associated.

As used herein, the term “polypeptide” refers broadly to a polymer of two or more amino acids joined together by peptide bonds. The term “polypeptide” also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide, enzyme, and protein are all included within the definition of polypeptide and these teams are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded RNA and DNA. A polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques. A polynucleotide can be linear or circular in topology. A polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment. A polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.

As used herein, the terms “coding region” and “coding sequence” are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5′ end and a translation stop codon at its 3′ end. A “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term “operably linked” refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner. A regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

A polynucleotide, such as a polynucleotide that includes a coding region, may include heterologous nucleotides on one or both sides of the polynucleotide. As used herein, “heterologous nucleotides” and “heterologous polynucleotides” are used interchangeably and refer to nucleotides that are not normally present flanking a polynucleotide, such as a coding region, that is present in a wild-type cell. For instance, a coding region present in a wild-type microbe and encoding a Cmr1 polypeptide described herein is flanked by homologous sequences, and any other nucleotide sequence flanking the coding region is considered to be heterologous. Examples of heterologous nucleotides include, but are not limited to regulatory sequences. Typically, heterologous nucleotides are present in a polynucleotide disclosed herein through the use of standard genetic and/or recombinant methodologies well known to one skilled in the art. A polynucleotide disclosed herein may be included hi a suitable vector.

As used herein, an “exogenous polynucleotide” refers to a polynucleotide that is not normally or naturally found in a microbe. As used herein, the term “endogenous polynucleotide” refers to a polynucleotide that is normally or naturally found in a microbe, e.g., genomic DNA is endogenous. An “endogenous polynucleotide” is also referred to as a “native polynucleotide.”

The terms “complement” and “complementary” as used herein, refer to the ability of two single stranded polynucleotides to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. The terms “substantial complement,” “substantially complementary,” and “substantial complementarity” as used herein, refer to a polynucleotide that is capable of selectively hybridizing to a specified polynucleotide under stringent hybridization conditions. Stringent hybridization for RNA molecules can take place under a number of pH, salt and temperature conditions. The pH can vary from 6 to 9, and may be 7 to 8, such as 7.4, and can be attained using 20 mM to 40 mM HEPES, such as 30 mM HEPES. The salt concentration can vary from 90 mM to 110 mM potassium acetate, such as 100 mM potassium acetate, and 1 mM to 3 mM magnesium acetate, such as 2 mM magnesium acetate. The temperature of the hybridization reaction may be incubation at 37° C. after a brief incubation at a higher temperature, such as 95° C. Thus, a polynucleotide is typically “substantially complementary” to a second polynucleotide if hybridization occurs between the polynucleotide and the second polynucleotide.

As used herein, “identity” refers to sequence similarity between two polypeptides or two polynucleotides. The sequence similarity between two polypeptides may be determined by aligning the residues of the two polypeptides (e.g., a candidate amino acid sequence and a reference amino acid sequence, such as SEQ ID NO:4, 6, 8, 10, 12, 14, or 16) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence analysis techniques such as the BESTFIT or GAP algorithm in the GCG package (Madison Wis.), or the Blastp program of the BLAST 2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, sequence similarity between two amino acid sequences is determined using the Blastp program of the BLAST 2 search algorithm. Preferably, the default values for all BLAST 2 search parameters are used, including matrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, sequence similarity is referred to as “identities.”

The sequence similarity between two polynucleotides may be determined by aligning the residues of the two polynucleotides (e.g., a candidate nucleotide sequence and a reference nucleotide sequence, such as SEQ ID NO:3, 5, 7, 9, 11, 13, or 15) to optimize the number of identical nucleotides along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of shared nucleotides, although the nucleotides in each sequence must nonetheless remain in their proper order. The sequence similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity. Sequence similarity may be determined, for example, using sequence techniques such as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector 4.5 (Kodak/IBI software package) or other suitable sequence analysis programs or methods known in the art. Preferably, sequence similarity between two nucleotide sequences is determined using the Blastn program of the BLAST 2 search algorithm, as described by Tatusova, et al. (1999, FEMS Microbiol Lett., 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health. Preferably, the default values for all BLAST 2 search parameters are used, including reward for match=1, penalty for mismatch=−2, open gap penalty=5, extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, and optionally, filter on. In the comparison of two nucleotide sequences using the BLAST search algorithm, sequence similarity is referred to as “identities.”

As used herein, “genetically modified microbe” refers to a microbe which has been altered by a person. A genetically modified microbe includes a microbe into which has been introduced a polynucleotide, such as an exogenous polynucleotide. Genetically modified microbe also refers to a microbe that has been genetically manipulated such that endogenous nucleotides have been altered to include a mutation, such as a deletion, an insertion, a transition, a transversion, or a combination thereof. For instance, an endogenous coding region could be deleted. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified microbe is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region.

Conditions that are “suitable” for an event to occur, such as cleavage of a polynucleotide, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.

As used herein, “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes. The term “in vivo” refers to the natural environment (e.g., a cell, including a genetically modified microbe) and to processes or reactions that occur within a natural environment.

As used herein “prokaryotic microbe” and “microbe” are used interchangeably and refer to members of the domains Bacteria and Archaea.

The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. P. furiosus CRISPR loci and distribution of cloned psiRNAs. The seven P. furiosus CRISPR loci are illustrated. The psiRNA numbers associated with each locus are indicated above. Each psiRNA is represented by a box. Shaded psiRNAs were cloned at least once in this work and the number of clones isolated is indicated below the shaded box. The genome coordinates of each P. furiosus CRISPR locus are as follows: 1: 27091-30618; 2: 260714-262113; 4: 312405-313931; 5: 623119-625176; 6: 695937-698992; 7: 1064076-1065543; 8: 1091089-1091857.

FIG. 2. Northern analysis of RNAs containing CRISPR repeat and psiRNA 4.02 sequences. A and B) Northern blots were performed with 10 μg total RNA using a degenerate oligonucleotide probe designed to detect the repeat sequences for CRISPR loci 1, 5, and 6 (A) or a probe for psiRNA 4.02 sequence (B). Radiolabeled RNA marker sizes are indicated (M). The positions of the initial transcript and large intermediates, 2× intermediate, 1× intermediate and primary psiRNA described in the text and FIG. 4C are indicated. C) Proposed psiRNA biogenesis pathway. The CRISPR locus is transcribed from a start site within the leader sequence to produce an initial transcript that includes a portion of the leader and the alternating psiRNA and repeat sequences. The initial transcript is cleaved within the repeats to produce intermediates. The endonucleolytic cleavage site may be asymmetrically located within the repeat. The 2× intermediate and 1× intermediates are illustrated. Our results indicate that the 1× intermediate is further processed by an exonuclease to remove most of the repeat sequence, resulting in a primary psiRNA species that contains 5-10 nucleotides of the repeat sequence.

FIG. 3. Northern analysis of RNAs from the seven P. furiosus CRISPR loci. A and B) Northern analysis was performed with 10 μg of total RNA using probes to detect sense (A) (transcribed from the leader sequence) or anti-sense (B) psiRNA or repeat sequence-containing RNAs as indicated. Lanes are approximately aligned on the basis of adjacent marker lanes (not shown except for repeat 1, 5, 6 and psiRNA 8.01 lanes). Dots located to the left of lanes indicate the primary psiRNA species. 1× and 2× intermediates detected by repeat probes are indicated.

FIG. 4. CRISPR RNA-protein complexes in fractionated P. furiosus cell extract. A) P. furiosus S100 cell extract was separated by DEAE anion exchange chromatography and CRISPR RNAs present in the fractions were examined by Northern analysis using probes against repeat 1, 5, 6, and psiRNAs 7.01 and 4.02 as indicated. Unfractionated extract (T) was co-analyzed for reference. Positions of 1× intermediate, 2× intermediate and primary psiRNA, peaks A-D (see Example 1), and markers are indicated. B) Fractions corresponding to peaks A (left), B (center) and C (right) were analyzed by non-denaturing gel electrophoresis and Northern blotting using a probe against psiRNA 7.01. For comparison, proteins were extracted from a portion of each sample and analysis of the RNAs with (+) and without (−) proteins is shown. The positions of the RNAs (−proteins) and potential RNPs (+proteins) on the native gel are indicated.

FIG. 5. Identification of a ribonucleoprotein complex containing psiRNAs and Cas proteins. A) psiRNP purification scheme. Letters indicate the location of corresponding data within the Figure. B) psiRNA (top panel) and total RNA (bottom panel) profiles across the initial Q-sepharose anion exchange fractions and an unfractionated sample (total). Northern analysis (top panel) was performed for P. furiosus psiRNA 7.01. The positions of the mature psiRNAs and 1× intermediate RNA (Hale et al., 2008, RNA 14, 2572-2579) are indicated. The lower panel shows all RNAs detected by SYBR Gold staining. The peak fraction is indicated by an arrow in each panel. C) psiRNA (Northern analysis of psiRNA 7.01, left panel) and total RNA (SYBR staining, right panel) profiles across the S-sepharose cation exchange fractions and starting material (load). The peak fraction is indicated by an arrow in each panel. D) Native gel Northern analysis of the psiRNP. The peak S-sepharose fraction (arrow, C) was fractionated by native gel electrophoresis and analyzed by Northern blotting for psiRNA 7.01. RNA extracted from the same fraction was co-analyzed. The position of the psiRNP is indicated. E) Cas proteins identified by tandem mass spectrometry. The isolated psiRNP (D) was subject to in-gel trypsin digestion and tandem mass spectrometry. Sequence coverage and the number of unique peptides for Cas proteins identified with 99% confidence are shown. P. furiosus cas gene names are as given (Haft et al., 2005, PLoS Comput Biol, 1:e60), and proposed functions are as predicted (Haft et al., 2005, PLoS Comput Biol, 1:e60; Makarova et al., 2006, Biol. Direct 1:7). F) Genome organization of predicted P. furiosus cas genes. Operon organization and COG assignments were adapted from NCBI database. Core cas genes (cas) and Cas module-RAMP (cmr), Cas subtype Apern (csa) and Cas subtype Tneap (cst) genes are indicated. Proteins identified by mass spectrometry are indicated in black.

FIG. 6. psiRNA species in the RNP contain a common 5′ sequence element and distinct 3′ termini. A) Sequence analysis of RNAs associated with the complex. RNA species present in the S-sepharose fraction (visualized by SYBR Gold staining) are shown in left panel. RNAs in the upper and lower bands were isolated, cloned, and sequenced. Graphs show the percentage of sequenced RNAs with 5′ ends located at specific positions within the repeat sequence (black), and with indicated numbers of guide sequence nucleotides downstream of the repeat sequence (orange). The average guide sequence is 37 nucleotides in P. furiosus. A consensus for each psiRNA species is diagrammed under each graph. The 8-nucleotide repeat sequence found at the 5′ end of the majority of the psiRNAs is indicated as the psi-tag. B) Model for biogenesis of the two psiRNA species in P. furiosus. CRISPR locus transcripts containing alternating repeat (R, black segments) and guide (G, shaded segments) elements are cleaved at a specific site within the repeat by the Cas6 endoribonuclease (Carte et al., 2008, Genes Dev, 22:3489-3496), ultimately producing 1× intermediate RNAs that contain a full invader-targeting sequence flanked on both sides by segments of the repeat. The mature RNAs retain the 5′ end repeat sequence (psi-tag). Uncharacterized 3′ end processing of the 1× intermediate by endo- and/or exo-nucleases forms the two major mature psiRNAs: a 45-nucleotide species that contains the 8-nucleotide psi-tag and a full guide sequence, and a 39-nucleotide species that contains a shorter 31-nucleotide guide sequence. C) Deep sequencing of small RNAs from P. furiosus confirms the presence of the psi-tag. The 5′ ends of the sequenced psiRNAs are graphed as in A. The number of total clones analyzed (n) is indicated in the graphs of panels A and C.

FIG. 7. Specific cleavage of complementary target RNAs. The indicated 5′ endlabeled substrates were incubated in the presence (+) or absence (−) of the psiRNP (FIG. 1C). Products were resolved by denaturing gel electrophoresis. In the upper panel, the primary cleavage products are indicated by arrows. For each lane, the arrow at the higher molecular weight corresponds to the site of cleavage indicated by the vertical lines in the substrate sequences shown in the lower panel and labeled Site 2, and the arrow at the lower molecular weight corresponds to the site of cleavage indicated by the vertical lines in the substrate sequences shown in the lower panel and labeled Site 1. The sizes of RNA markers (M) are indicated. “Target” substrates (panels 1, 3, 4, 5, 7) contain regions of perfect complementarity to the guide sequence of the indicated P. furiosus psiRNA. Grey bars demarcate the guide sequences in the lower panel. “+ext” substrates (panels 1, 2, 3, 4, 7) contain 5′ and 3′ polyA extensions. In panel 4, a synthetic psiRNA (sequence shown in grey) was pre-annealed to the 7.01 target RNA+ext. Panel 2 shows a reverse target sequence substrate and panel 6 shows an antisense (AS) target substrate. Panel 3 shows a DNA substrate; all other substrates are RNA. Panel 8 shows unrelated RNA sR2.

FIG. 8. Cleavages occurs 14 nucleotides from the 3′ ends of the psiRNAs. A) The indicated 5′ end-labeled (*) substrates were incubated in the presence (+) or absence (−) of the psiRNP (FIG. 1C). The substrates were full-length 7.01 target RNA (F.L.), and the indicated truncations as diagramed in the lower panel. As in FIG. 3, the locations of observed cleavages at sites 1 and 2 intermediate RNAs that contain a full invader-targeting sequence flanked on both sides by segments of the repeat. The mature RNAs retain the 5′ end repeat sequence (psi-tag). Uncharacterized 3′ end processing of the 1× intermediate by endo- and/or exo-nucleases forms the two major mature psiRNAs: a 45-nucleotide species that contains the 8-nucleotide psi-tag and a full guide sequence, and a 39-nucleotide species that contains a shorter 31-nucleotide guide sequence. C) Deep sequencing of small RNAs from P. furiosus confirms the presence of the psi-tag. The 5′ ends of the sequenced psiRNAs are graphed as in A. The number of total clones analyzed (n) is indicated in the graphs of panels A and C.

FIG. 7. Specific cleavage of complementary target RNAs. The indicated 5′ endlabeled substrates were incubated in the presence (+) or absence (−) of the psiRNP (FIG. 1C). Products were resolved by denaturing gel electrophoresis. In the upper panel, the primary cleavage products are indicated by arrows. For each lane, the arrow at the higher molecular weight corresponds to the site of cleavage indicated by the vertical lines in the substrate sequences shown in the lower panel and labeled Site 2, and the arrow at the lower molecular weight corresponds to the site of cleavage indicated by the vertical lines in the substrate sequences shown in the lower panel and labeled Site 1. The sizes of RNA markers (M) are indicated. “Target” substrates (panel 1 (SEQ ID NO:260); panel 3 (SEQ ID NO:262); panel 4 (SEQ ID NO: 264); panel 5 (SEQ ID NO: 265); panel 7 (SEQ ID NO:267)) contain regions of perfect complementarity to the guide sequence of the indicated P. furiosus psiRNA. Grey bars demarcate the guide sequences in the lower panel. “+ext” substrates (panels 1, 2, 3, 4, 7) contain 5′ and 3′ polyA extensions. In panel 4, a synthetic psiRNA (SEQ ID NO: 263; sequence shown in grey) was pre-annealed to the 7.01 target RNA+ext. Panel 2 shows a reverse target sequence substrate (SEQ ID NO: 261) and panel 6 shows an antisense (AS) target substrate (SEQ ID NO: 266). Panel 3 shows a DNA substrate; all other substrates are RNA. Panel 8 shows unrelated RNA sR2 (SEQ ID NO:268).

FIG. 8. Cleavages occurs 14 nucleotides from the 3′ ends of the psiRNAs. A) The indicated 5′ end-labeled (*) substrates were incubated in the presence (+) or absence (−) of the psiRNP (FIG. 1C). The substrates were full-length 7.01 target RNA (F.L.), and the indicated truncations as diagramed in the lower panel. As in FIG. 3, the locations of observed cleavages at sites 1 and 2 are indicated on the full-length target and truncated RNAs (lower panel) and the corresponding cleavage products are indicated with arrows in the upper panels. Question mark in the lower panel indicates cleavage that could not be assessed. B) Model for cleavage at two sites directed by two psiRNAs. The 45-nucleotide psiRNA species guides cleavage at site 1 and the 39-nucleotide psiRNA guides cleavage at site 2 on each of the substrate RNAs as indicated. In both cases, cleavage occurs 14 nucleotides from the 3′ end of the psiRNA. Observed products are indicated with regular type and shaded bars, and products not observed are indicated in gray type, and correspond to products in FIGS. 3 and 4A.

FIG. 9. Target RNA cleavage requires five Cmr proteins and a single psiRNA species. A) 5′ end-labeled 7.01 target RNA was incubated in the absence of added psiRNAs or proteins, in the presence of synthetic psiRNAs or purified recombinant P. furiosus Cmr proteins (R) or in the presence of purified native psiRNPs (N) as indicated. The synthetic psiRNAs were the 45- and 39-nucleotide forms of psiRNA 7.01. The six added recombinant Cmr proteins were P. furiosus Cmr1-1, Cmr2, Cmr3, Cmr4, Cmr5 and Cmr6. Products were resolved by denaturing gel electrophoresis. The products corresponding to cleavage at site 1 and site 2 are indicated by lower and upper arrows, respectively. The sizes of RNA markers (M) are indicated. B) The 7.01 target, along with the 7.01 psiRNAs (both 39- and 45-nucleotide species, in the presence of Cmr1-6 (all), and in the absence (−) of the indicated Cmr protein. The locations of the cleavage products are indicated as above. C) The cleavage activity of the native psiRNP(N) is compared to the activity of the recombinant psiRNP(R) in the presence of either the 45-nucleotide 7.01 psiRNA, the 39-nucleotide 7.01 psiRNA, or both psiRNAs. Cleavage sites are indicated as above.

FIG. 10. Model for the function of psiRNA-Cmr protein complexes in silencing molecular invaders. Based on the results of this study, a psiRNA with a conserved 5′ sequence element derived from the CRISPR repeat (psi-tag) and a region of invadertargeting sequence assembles with six Cas module-RAMP proteins (Cmr1-6). The assembled psiRNP interacts with an invader RNA (e.g., viral mRNA) through base pairing between the psiRNA and invader RNA, positioning the region of the RNA-RNA duplex 14 nucleotides from the 3′ end of the psiRNA in proximity to the active site (star) of the enzyme. In P. furiosus, there are two prominent size forms of psiRNAs with different 3′ ends that guide cleavage of viral mRNAs at two distinct sites. There are also two Cmr1 proteins in P. furiosus that are both found in purified preparations and likely function redundantly.

FIG. 11. Deep sequencing of small RNAs from P. furiosus, Solfolobus solfataricus and Sulfolobus tokodaii confirms the presence of the psi-tag. The 5′ ends of the sequenced psiRNAs are graphed as in FIG. 6A. The number of total clones analyzed (n) is indicated.

FIG. 12. The psi-tag is required for cleavage of complimentary sequences by the Cmr complex. A psiRNA with the wildtype tag (AUUGAAAG, wt) efficiently guides cleavage of a complementary RNA in the presence of Cmr1-6 (+proteins). The same psiRNA that is lacking the tag sequence (-tag) is unable to guide cleavage of the target. Mutating the tag sequence to its complement, UAACUUUC (comp. tag), also inactivates the complex. This indicates that the tag sequence, AUUGAAAG, is required for cleavage of a complementary RNA by the Cmr proteins.

FIG. 13. The psi-tag allows for rational design of psiRNAs that cleave novel targets. psiRNAs were created to target two non-natural sequences. The first (exogenous) psiRNA targets an unrelated sequence, and the second (bla) psiRNA targets the first 37 nucleotides of the β-lactamase, or bla, mRNA, which confers antibiotic resistance in bacteria. The psiRNAs were created by adding the tag sequence, AUUGAAAG, to 37 nucleotides of guide sequence, which is complementary to the targeted sequence. The psiRNAs were incubated with the Cmr proteins and allowed to cleave both of the targets. The exogenous psiRNA was able to guide cleavage of the complementary sequence (exogenous target), but not the bla target, and the bla psiRNA was able to guide cleavage of the bla message, but not the exogenous sequence. These results indicate that addition of the psi-tag to any sequence will allow for specific cleavage of the complementary sequence by the Cmr proteins.

FIG. 14. Amino acid sequences of Cmr polypeptides and nucleotide sequences encoding the polypeptides.

FIG. 15. Alignments between Cmr polypeptide regions and domains of hidden Markov models present in the TIGRFAM database of protein families.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes aspects that represent an advance in the art of inactivating polynucleotides, preferably cleaving polynucleotides in a prokaryotic microbe. A CRISPR locus of a prokaryotic microbe includes, from 5′ to 3′, a repeat followed immediately by a spacer (referred to herein as a “repeat-spacer unit”). Typically, a CRISPR locus includes multiple repeat-spacer units. In a CRISPR locus, each repeat is nearly identical (Barrangou et al., U.S. Published Patent Application 2008/0124725). In contrast to the repeats, each spacer of a CRISPR locus is typically a different nucleotide sequence.

As described herein, a CRISPR locus is transcribed in a microbe and subsequently processed to produce psiRNAs, and the psiRNAs are proposed to be primary agents in guiding the inactivation of target polynucleotides. A target polynucleotide is inactivated when it is no longer able to carry out its biological function. For instance, an mRNA is no longer able to carry out its biological function when it no longer encodes an active polypeptide. Inactivation may occur by degradation, such as by cleavage. As described in the examples below, the first 5 to 10 nucleotides, preferably, the first 8 nucleotides, of a psiRNA may be derived from the repeat of a repeat-spacer unit. The remaining nucleotides of a psiRNA may be derived from the spacer of the repeat-spacer unit. Thus, for any psiRNA derived from a repeat-spacer unit, the nucleotides derived from the repeat are those nucleotides immediately upstream of the spacer, and the nucleotides derived from the spacer are those nucleotides immediately downstream of the repeat.

Accordingly, an aspect of the present invention includes polynucleotides having a 5′ region and a 3′ region. Such polynucleotides may be referred to herein as a “psiRNA,” and may be enriched or isolated. While the term psiRNA suggests the nucleotides are ribonucleotides, polynucleotides described herein also include the corresponding deoxyribonucleotide sequence, and the RNA and DNA complements thereof. It should be understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to

FIG. 15. Alignments between Cmr polypeptide regions and domains of hidden Markov models present in the TIGRFAM database of protein families. Cmr1-1 (amino acids 6 to 161 or 8 to 164 of SEQ ID NO:4), domain present in PF03787 (SEQ ID NO:269), domain present in TIGR01894 (SEQ ID NO:270), Cmr2 (amino acids 220 to 739 of SEQ ID NO:8), domain present in TIGR02577 (SEQ ID NO:271), Cmr3 (amino acids 4 to 321 of SEQ ID NO:10), domain present in TIGR01888 (SEQ ID NO:272), Cmr4 (amino acids 4 to 280 or 6 to 280 of SEQ ID NO:12), domain present in PF03787 (SEQ ID NO:269), domain present in TIGR02580 (SEQ ID NO:273), Cmr5 (amino acids 15 to 161 of SEQ ID NO:14), domain present in TIGR01881 (SEQ ID NO:274), Cmr6 (amino acids 111 to 337 of SEQ ID NO:16), domain present in TIGR01898 (SEQ ID NO:275), domain present in PF03787 (SEQ ID NO:269).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes aspects that represent an advance in the art of inactivating polynucleotides, preferably cleaving polynucleotides in a prokaryotic microbe. A CRISPR locus of a prokaryotic microbe includes, from 5′ to 3′, a repeat followed immediately by a spacer (referred to herein as a “repeat-spacer unit”). Typically, a CRISPR locus includes multiple repeat-spacer units. In a CRISPR locus, each repeat is nearly identical (Barrangou et al., U.S. Published Patent Application 2008/0124725). In contrast to the repeats, each spacer of a CRISPR locus is typically a different nucleotide sequence.

As described herein, a CRISPR locus is transcribed in a microbe and subsequently processed to produce psiRNAs, and the psiRNAs are proposed to be primary agents in guiding the inactivation of target polynucleotides. A target polynucleotide is inactivated when it is no longer able to carry out its biological function. For instance, an mRNA is no longer able to carry out its biological function when it no longer encodes an active polypeptide. Inactivation may occur by degradation, such as by cleavage. As described in the examples below, the first 5 to 10 nucleotides, preferably, the first 8 nucleotides, of a psiRNA may be derived from the repeat of a repeat-spacer unit. The remaining nucleotides of a psiRNA may be derived from the spacer of the repeat-spacer unit. Thus, for any psiRNA derived from a repeat-spacer unit, the nucleotides derived from the repeat are those nucleotides immediately upstream of the spacer, and the nucleotides derived from the spacer are those nucleotides immediately downstream of the repeat.

Accordingly, an aspect of the present invention includes polynucleotides having a 5′ region and a 3′ region. Such polynucleotides may be referred to herein as a “psiRNA,” and may be enriched or isolated. While the term psiRNA suggests the nucleotides are ribonucleotides, polynucleotides described herein also include the corresponding deoxyribonucleotide sequence, and the RNA and DNA complements thereof. It should be understood that the sequences disclosed herein as DNA sequences can be converted from a DNA sequence to an RNA sequence by replacing each thymidine nucleotide with a uracil nucleotide. The 5′ region of a polynucleotide, also referred to herein as a “tag” or some variant thereof, such as “psiRNA-tag” or “psi-tag,” includes at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides. In some aspects, a psi-tag is 5, 6, 7, 8, 9, or 10 nucleotides. The nucleotide sequence of a psiRNA-tag is identical to or has sequence similarity with the nucleotide sequence of a repeat (from a CRISPR locus) that is immediately upstream of a spacer.

The nucleotide sequence of a psiRNA-tag of a polynucleotide described herein is identical to or has sequence similarity with the 5 to 10 nucleotides of a repeat from a CRISPR locus that is immediately upstream of a spacer and present in a microbe that contains a CRISPR locus. psiRNA-tag nucleotide sequences that can be present in a polynucleotide of the present invention can easily be identified in any microbe that includes a CRISPR locus. For instance, the genomic sequences of many microbes are known, and the location of CRISPR loci in these microbes is often known, or can easily be located using routine bioinformatic methods known in the art. For instance, Edgar (BMC Bioinformatics, 2007, 8:18) describes a computer program specifically designed for the identification and analysis of CRISPR repeats, and includes a list of predicted repeats based on 346 prokaryotic genomes (see Edgar, Supplementary Table 1). Grissa et al. (BMC Bioinformatics, 2007, 8:172, and Nucl. Acids Res., 2007, 35 (Web Server issue):W52-W57) describe a computer program which identifies CRISPRs from genomic sequences, extracts the repeat and spacer sequences, and constructs a database which is automatically updated monthly using newly released genome sequences. Thus, the nucleotide sequence of a repeat that is immediately upstream of a spacer in a CRISPR locus can be determined by the skilled person using routine methods.

The psiRNAs of the present invention include 5′ nucleotide sequences identical to or having sequence similarity with the nucleotide sequence of a repeat from a CRISPR locus that is immediately upstream of a spacer; however, due to the ease of identifying repeats from CRISPR loci, the psiRNAs of the present invention are not intended to be limited to any specific CRISPR locus repeat sequence. Examples of psiRNA-tag nucleotide sequences include sequences disclosed in Kunin et al. (Genome Biol., 2007, 8:R61.1-R61.7), Godde and Bickerton (J. Mol. Evol., 62:718-729), and Edgar (BMC Bioinformatics, 2007, 8:18). An example of a psiRNA-tag of a polynucleotide of the present invention is 5′ ATTGAAAG (or 5′ AUUGAAAG when the polynucleotide is RNA) or 5′ ATTGAAAC (or 5′ AUUGAAAC when the polynucleotide is RNA), and polynucleotides having sequence similarity with ATTGAAAG or ATTGAAAC.

In some aspects a nucleotide sequence of a psiRNA-tag of a polynucleotide of the present invention may further include additional nucleotides that are identical or have sequence similarity to the other nucleotides of a repeat from a CRISPR locus that is immediately upstream of a spacer. Thus, in some embodiments, a psiRNA-tag may include nucleotides that are identical or have sequence similarity with an entire repeat, or a subset of nucleotides present in the repeat. Typically, if a psiRNA-tag includes nucleotides identical or have sequence similarity with a subset of nucleotides present in a repeat, the subset of nucleotides present in the psiRNA-tag are typically from the 3′ end of the repeat. For instance, an example of a repeat present in Pyrococcus furiosus is GTTCCAATAAGACTAAAATAGAATTGAAAG (SEQ ID NO:276). Examples of psiRNA-tags of a polynucleotide of the present invention include having a subset of nucleotides present in this repeat are, but are not limited to, TTCCAATAAGACTAAAATAGAATTGAAAG (nucleotides 2-30 of SEQ ID NO:276) TCCAATAAGACTAAAATAGAATTGAAAG U (nucleotides 3-30 of SEQ ID NO:276), and CCAATAAGACTAAAATAGAATTGAAAG (nucleotides 4-30 of SEQ ID NO:276). Thus, in some embodiments, a psiRNA-tag of a polynucleotide of the present invention includes at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, and so on up to inclusion of nucleotides from an entire repeat. In other embodiments, a psiRNA of the present invention does not include additional nucleotides located upstream of the psiRNA-tag.

The 3′ region of a psiRNA of the present invention is immediately downstream of the 5′ region and may be referred to herein as a spacer sequence or a guide sequence. Without intending to be limited by theory, the guide sequence directs a psiRNA of the invention to identify a specific polynucleotide, referred to herein as a target polynucleotide, optionally resulting in inactivation of the target polynucleotide. The target polynucleotide may be DNA or RNA. In some aspects, the target polynucleotide is preferably RNA. Accordingly, in one aspect the guide sequence of a psiRNA of the present invention may be either substantially complementary or complementary to the target polynucleotide. The 3′ region of a psiRNA includes a number of nucleotides that is an integer greater than 17 (e.g., at least 18, at least 19, at least 20). In some aspects, the 3′ region of a psiRNA may be an integer less than 76 (e.g., no greater than 75, no greater than 74, no greater than 73, and so on). In some embodiments, the 3′ region of a psiRNA is at least 28, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, or at least 39 nucleotides. Thus, a psiRNA of the present invention may be at least 23 nucleotides in length, or greater. In some aspects, a psiRNA of the present invention may be 39 nucleotides or 45 nucleotides in length, for instance, an 8 nucleotide tag and a guide of 31 or 37 nucleotides.

The 3′ region of a psiRNA may be any nucleotide sequence. As discussed below, one aspect of the present invention includes methods for cleaving a specific target polynucleotide at a specific location, e.g., using a psiRNA as a restriction endonuclease that cleaves a polynucleotide, such as an RNA polynucleotide. Thus, in one aspect, since the 3′ region of a psiRNA is substantially complementary or complementary to the target polynucleotide, the sequence of the target polynucleotide will dictate the nucleotide sequence of a psiRNA 3′ region. Specific examples of nucleotide sequences that can be present in a psiRNA 3′ region are described hereinbelow.

Also provided herein are polypeptides that have endonuclease activity, for instance, endoribonuclease activity, when used together. The polypeptides are referred to herein as Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

Examples of Cmr1 polypeptides are depicted at Genbank Accession No. AAL81254 (SEQ ID NO:4), Genbank Accession No. AAL80476 (SEQ ID NO:6), and FIG. 14. The Cmr1 polypeptide having SEQ ID NO:6 is expected to have endonuclease activity, for instance, endoribonuclease activity, under the conditions described herein. Other examples of Cmr1 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:6. A Cmr1 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:4 or SEQ ID NO:6 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr2, Cmr3, Cmr4, Cmr5 and Cmr6 polypeptides having amino acid sequences SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

Examples of Cmr2 polypeptides are depicted at Genbank Accession No. AAL81253 (SEQ ID NO:8) and FIG. 14. Other examples of Cmr2 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:8. A Cmr2 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:8 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr3, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

Examples of Cmr3 polypeptides are depicted at Genbank Accession No. AAL81252 (SEQ ID NO:10) and FIG. 14. Other examples of Cmr3 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:10. A Cmr3 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:10 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr4, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

Examples of Cmr4 polypeptides are depicted at Genbank Accession No. AAL81250 (SEQ ID NO:12) and FIG. 14. Other examples of Cmr4 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:12. A Cmr4 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:12 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, Cmr5, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, and SEQ ID NO:16.

Examples of Cmr5 polypeptides are depicted at Genbank Accession No. AAL81249 (SEQ ID NO:14) and FIG. 14. Other examples of Cmr5 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:14. A Cmr5 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:14 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, Cmr4, and Cmr6 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:16.

Examples of Cmr6 polypeptides are depicted at Genbank Accession No. AAL81248 (SEQ ID NO:16) and FIG. 14. Other examples of Cmr6 polypeptides provided herein include those having sequence similarity with the amino acid sequence of SEQ ID NO:16. A Cmr6 polypeptide having sequence similarity with the amino acid sequence depicted at SEQ ID NO:16 has endonuclease activity, for instance, endoribonuclease activity, when incubated with a psiRNA, a target polynucleotide, and Cmr1, Cmr2, Cmr3, and Cmr4 polypeptides having amino acid sequences SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14.

A composition including a Cmr1 polypeptide chosen from SEQ ID NO:4 and SEQ ID NO:6, preferably SEQ ID NO:4, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, and a Cmr6 polypeptide has endonuclease activity. Optionally, a composition also includes a Cmr5 polypeptide. The endonuclease activity of a composition of five, optionally six, of the polypeptides described herein acts to inactivate a target polynucleotide. Inactivation may be by

cleavage of a target polynucleotide, preferably at a specific site. When determining whether a composition of five, optionally six, of the polypeptides described herein have endonuclease activity, a suitable psiRNA is 5′-AUUGAAAGUUGUAGUAUGCGGUCCUUGCGGCUGAGAGCACUUCAG (where the first 8 nucleotides are the 5′ region and the remaining nucleotides are the 3′ region, SEQ ID NO:17) and a suitable target polynucleotide is 5′-CUGAAGUGCUCUCAA↓GCCGCAAGGACCGCAUACUACAA (SEQ ID NO:18), where the target polynucleotide is optionally cleaved, for example, between the nucleotides as defined by the arrow. Suitable conditions for cleavage of this polynucleotide by a composition of the polypeptides described herein include those described in Example 2.

The amino acid sequence of a polypeptide having sequence similarity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16 may include conservative substitutions of amino acids present in an amino acid sequence. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) may generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Val, Leu, and Ile (representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: Glu, Asp, Asn and Gln (carboxyl or amide group-containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gln (representing amide side chains). The classes are not limited to naturally occurring amino acids, but also include artificial amino acids, such as beta or gamma amino acids and those containing non-natural side chains, and/or other similar monomers such as hydroxyacids.

Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990, Science, 247:1306-1310), wherein the authors indicate proteins are surprisingly tolerant of amino acid substitutions. For example, Bowie et al. disclose that there are two main approaches for studying the tolerance of a polypeptide sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As stated by the authors, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein.

Cmr2 polypeptides typically include polymerase/nuclease domains (see Makarova et al., 2002, Nucleic Acids Res., 30:482-496, and Makarova et al., 2006, Biol. Direct 1:7). Cmr1, Cmr3, Cmr4, and Cmr6 are members of the RAMP superfamily (Markova et al., 2002, Nucl. Acids Res., 30:482-496), and may include the characteristics of polypeptides that are members of that superfamily. Cmr1 polypeptides may include domains present in the TIGRFAM database at accession numbers PF03787 and TIGR01894, as shown in FIG. 15. The TIGRFAM database includes families of polypeptides for which function is conserved (Haft et al., Nucl. Acids Res., 2003, 31:371-373, Bateman and Haft, 2002, Briefings Bioinformatics, 3:236-245, and Haft et al., 2005, PLoS Computational Biol., 1(6):e60). Cmr2 polypeptides may include domains present in the TIGRFAM database at accession number TIGR02577, as shown in FIG. 15. Cmr3 polypeptides may include domains present in the TIGRFAM database at accession number TIGR01888, as shown in FIG. 15. Cmr4 polypeptides may include domains present in the TIGRFAM database at accession numbers TIGR02580 and PF03787, as shown in FIG. 15. Cmr5 polypeptides may include domains present in the TIGRFAM database at accession numbers TIGR01881, as shown in FIG. 15. Cmr6 polypeptides may include domains present in the TIGRFAM database at accession numbers TIGR01898 and PF03787, as shown in FIG. 15.

A Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polypeptide may be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods. A Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polypeptide may be isolated from a prokaryotic microbes having CRISPR loci. Examples of prokaryotic microbes with known whole genomic sequences containing coding regions expected to encode a polypeptide of the present invention include Archaeoglobus fulgidus DSM4304, Clostridium botulinum A ATCC 19397, Clostridium botulinum A Hall, Clostridium botulinum F Langeland, Clostridium botulinum Hall A Sanger, Deinococcus geothermalis DSM 11300, Methanosaeta thermophila PT, Methanosarcina acetivorans C2A, Myxococcus xanthus DK 1622, Pyrococcus furiosus DSM 3638, Rubrobacter xylanophilus DSM 9941, Sulfolobus solfataricus P2, Sulfolobus tokodaii strain 7, Synechococccus sp. OS Type A, Synechococccus sp. OS Type B prime, Syntrophus aciditrophicus SB, Thermoanaerobacter tengcongensis MB4(T), Thermobifida fusca YX, Thermotoga maritima MSB8, Thermus thermophilus HB27, Porphyromonas gingivalis W83, Methanopyrus kandleri AV19, Clostridium novyi NT, Methanococcus jannaschii DSM2661, Staphylococcus epidermidis RP62A, Sulfolobus acidocaldarius DSM 639, Methanococcus maripaludis C5, Methanocorpusculum labreanum Z, Methanosarcina barkeri fusaro, Prochlorococcus marinus str. AS9601, Campylobacter fetus subsp. fetus 82-40, Carboxydothermus hydrogenoformans Z-2901, Clostridium botulinum B1 strain Okra, Methanospirillum hungatei JF-1, Mycobacterium tuberculosis CDC1551, Nostoc sp. PCC 7120, Pyrobaculum islandicum DSM 4184, Pyrococcus horikoshii shinkaj OT3, Burkholderia cenocepacia H12424, Neorickettsia sennetsu Miyayama, Rhodospirillum rubrum ATCC 11170, Clostridium beijerinckii NCIMB 8052, Aquifex aeolicus VF5, Bacillus halodurans C-125, Thermus thermophilus HB8, Methanosarcina mazei Goel, Treponema pallidum Nichols, Baumannia cicadellinicola, Chlamydia muridarum strain Nigg, Clostridium perfringens ATCC13124, Methylibium petroleiphilum PM1, Mycoplasma capricolumn subsp capricolumn California kid ATCC 27343, Mycoplasma genitalium G-37, Clostridium phytofermentans ISDg, Methanobacterium thermoautotrophicum delta H, Methanothermobacter thermautotrophicus str. Delta H, Ehrlichia chaffeensis Arkansas, Campylobacter curvus 525.92, Clostridium perfringens 13, Clostridium perfringens SM101, Coxiella burnetii RSA 493, Desulfovibrio vulgaris Hildenborough, Enterococcus faecalis V583, Lactobacillus delbrueckii subsp. bulgaricus ATCC BAA-365, Leptospira interrogans Copenhageni Fiocruz L1-130, Leptospira interrogans serovar lai str. 56601, Listeria welshimeri serovar 6b str. SLCC5334, Mesoplasma florum L1, Methanococcoides burtonii DSM 6242, Prevotella intermedia 17, Pseudomonas aeruginosa PA01, Pyrococcus abyssi GE5, Staphylococcus epidermidis ATCC 12228, Streptococcus gordonii Challis NCTC7868, Streptococcus pneumoniae TIGR4, Streptomyces coelicolor A3(2), Sulfolobus islandicus filamentous virus (SIFV), Aeropyrum pernix K1, Anaplasma phagocytophilum HZ, Campylobacter concisus 13826, Campylobacter jejuni RM1221, Colwellia psychrerythraea 34H, Deinococcus radiodurans R1, Geobacter sulfurreducens PCA, Nitrosospira multiformis ATCC 25196, Prochlorococcus marinus MIT9313, Pseudomonas putida KT2440, Shewanella oneidensis MR-1, Shewanella sp. MR-4, Shewanella sp. MR-7, Streptococcus agalactiae 2603V/R, Streptococcus agalactiae A909, Synechococcus sp. CC9311, Treponema denticola ATCC 35405, Vibrio cholerae El Tor N16961, Vibrio fischeri ES114, Bacillus halodurans, Synechocystis sp. PCC 6803, Thermotoga maritima, Borrelia garinii PBi, Lactococcus lactis subsp. lactis μL1403, Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293, Mannheimia succiniciproducens MBEL55E, Mycobacterium bovis subsp. bovis AF2122/97, Mycobacterium tuberculosis H37Rv (lab strain), Nitrosomonas europaea ATCC 19718, Photorhabdus luminescens TT01, Picrophilus torridus DSM 9790, Pyrobaculum aerophilum μM2, Streptococcus thermophilus CNRZ1066, Streptococcus thermophilus LMG 18311, Thermoplasma volcanium GSS1, and Vibrio vulnificus YJO16.

Also provided herein are polynucleotides, including isolated polynucleotides, encoding a Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr 5, or a Cmr6 polypeptide. Cmr1 polynucleotides may have a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6. Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 polynucleotides may have a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, respectively. An example of the class of nucleotide sequences encoding such a polypeptide is the nucleotide sequence depicted at Genbank Accession Numbers as shown in Table 1.

TABLE 1 Genbank accession numbers and PF locus numbers for Cmr polypeptides and polynucleotides. Genbank nucleotide Genbank sequence (location SEQ ID protein SEQ ID PF in genome) NO: sequence NO: locus Cmr1 NC_003413 3 AAL81254 4 PF1130 (1081353-1080337) Cmr1 NC_003413 5 AAL80476 6 PF0352 (365628-365038) Cmr2 NC_003413 7 AAL81253 8 PF1129 (1080344-1077729) Cmr3 NC_003413 9 AAL81252 10 PF1128 (1077732-1076764) Cmr4 NC_003413 11 AAL81250 12 PF1126 (1075334-1074447) Cmr5 NC_003413 13 AAL81249 14 PF1125 (1074469-1073960) Cmr6 NC_003413 15 AAL81248 16 PF1124 (1073976-1072954) It should be understood that a polynucleotide encoding a Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polypeptide represented by the corresponding nucleotide sequence in Table 1 is not limited to that nucleotide sequence, but also includes the class of polynucleotides encoding such polypeptides as a result of the degeneracy of the genetic code. For example, the naturally occurring nucleotide sequence SEQ ID NO:3 is but one member of the class of nucleotide sequences encoding a polypeptide having the amino acid sequence SEQ ID NO:4. The class of nucleotide sequences encoding a selected polypeptide sequence is large but finite, and the nucleotide sequence of each member of the class may be readily determined by one skilled in the art by reference to the standard genetic code, wherein different nucleotide triplets (codons) are known to encode the same amino acid.

A polynucleotide encoding a polypeptide described herein may have sequence similarity with the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15. A Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polynucleotide may be isolated from a microbe having CRIPSR loci, such as, but not limited to, Pyrococcus furiosus, or may be produced using recombinant techniques, or chemically or enzymatically synthesized using routine methods. A Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polynucleotide may further include heterologous nucleotides flanking the open reading frame encoding a Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polypeptide. Typically, heterologous nucleotides may be at the 5′ end of the coding region, at the 3′ end of the coding region, or the combination thereof. The number of heterologous nucleotides may be, for instance, at least 10, at least 100, or at least 1000.

The present invention also includes fragments of the polypeptides described herein, and the polynucleotides encoding such fragments, such as SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, as well as those polypeptides having sequence similarity with the polypeptides. A polypeptide fragment may include a sequence of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 amino acid residues.

A polypeptide disclosed herein or a fragment thereof may be expressed as a fusion polypeptide that includes an additional amino acid sequence. For instance, the additional amino acid sequence may be useful for purification of the fusion polypeptide by affinity chromatography. Various methods are available for the addition of such affinity purification moieties to proteins. Representative examples may be found in Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma (U.S. Pat. No. 5,594,115). In another example, the additional amino acid sequence may be a carrier polypeptide. The carrier polypeptide may be used to increase the immunogenicity of the fusion polypeptide to increase production of antibodies that specifically bind to a polypeptide of the invention. The invention is not limited by the types of carrier polypeptides that may be used to create fusion polypeptides. Examples of carrier polypeptides include, but are not limited to, keyhole limpet hemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like.

A polynucleotide disclosed herein, such as a polynucleotide encoding a psiRNA or a polynucleotide encoding a polypeptide described herein, may be present in a vector. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide of the invention employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. The term vector includes, but is not limited to, plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. Typically, a vector is capable of replication in a microbial host, for instance, a fungus, such as S. cerevisiae, or a prokaryotic microbe, such as E. coli. Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. In some aspects, suitable host cells for cloning or expressing the vectors herein include eukaryotic cells. Suitable eukaryotic cells include fungi, such as S. cerevisiae and P. pastoris. In other aspects, suitable host cells for cloning or expressing the vectors herein include prokaryotic cells. Suitable prokaryotic cells include bacteria, such as gram-negative microbes, for example, E. coli. Other suitable prokaryotic cells include archeae, such as Haloferax volcanii. Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing polynucleotides into host cells.

Polynucleotides encoding the polypeptides disclosed herein may be obtained from microbes, for instance, members of the genus Pyrococcus, such as P. furiosus, or produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known. Likewise, polypeptides of the present invention may be obtained from microbes, or produced in vitro or in vivo.

An expression vector may optionally include a promoter that results in expression of an operably linked psiRNA. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3′ direction) coding region. Promoters present in prokaryotic microbes typically include two short sequences at −10 (often referred to as the Pribnow box, or the −10 element) and −35 positions (often referred to as the −35 element), or a short sequence at −30 (often referred to as a TATA box) located 5′ from the transcription start site, for bacterial and archael organisms, respectively. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to a host cell. psiRNA polynucleotides of the present invention do not encode a polypeptide, and expression of a psiRNA present in a vector results in a non-coding RNA. Thus, a vector including a psiRNA may also include a transcription start signal and/or a transcription terminator operably linked to the psiRNA, but a translation start signal and/or translation stop signal typically are not operably linked to a psiRNA. Promoters have been identified in many microbes and are known to the skilled person. Many computer algorithms have been developed to detect promoters in genomic sequences, and promoter prediction is a common element of many gene prediction methods. Thus, the skilled person can easily identify nucleotide sequences present in microbes that will function as promoters.

An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. A vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to a selective agent, such as an antibiotic, or it may confer compound-specific metabolism on the transformed cell. Examples of a marker sequence include, but are not limited to, sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, streptomycin, and neomycin. Another example of a marker that renders a cell resistant to a selective agent is 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA), an enzyme used for archaeal membrane lipid biosynthesis (Matsumi et al., J. Bacteriol., 2007, 189:2683-2691). Certain statins, such as mevinolin and its analog simvastatin, inhibit HMG-CoA reductase activity, and overexpression of HMG-CoA reductase can confer resistance to mevinolin and/or simvastatin. Yet another example of a marker is a nutritional marker. A nutritional marker is typically a coding region that, when mutated in a cell, confers on that cell a requirement for a particular compound. Cells containing such a mutation will not grow on defined medium that does not include the appropriate compound, and cells receiving a coding region that complements the mutation can grow on the defined medium in the absence of the compound. Examples of nutritional markers include, but are not limited to, coding regions encoding polypeptides in biosynthetic pathways, such as nucleic acid biosynthesis (e.g., biosynthesis of uracil), amino acid biosynthesis (e.g., biosynthesis of histidine and tryptophan), vitamin biosynthesis (e.g., biosynthesis of thiamine), carbohydrate metabolism (e.g., metabolism of cellobiose), and the like.

Polypeptides and fragments thereof useful in the present invention may be produced using recombinant DNA techniques, such as an expression vector present in a cell. Such methods are routine and known in the art. The polypeptides and fragments thereof may also be synthesized in vitro, e.g., by solid phase peptide synthetic methods. The solid phase peptide synthetic methods are routine and known in the art. A polypeptide produced using recombinant techniques or by solid phase peptide synthetic methods may be further purified by routine methods, such as fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or on an anion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75, or ligand affinity.

Polypeptides useful in the methods described herein, such as the polypeptides described herein and other polypeptides belonging to the Cmr family, may be obtained from a microbe that has a CRISPR locus. Examples of such microbes are listed hereinabove. Methods for obtaining polypeptides useful in the methods described herein may include ion exchange chromatography of cellular extracts. Typically, obtaining polypeptides includes conditions that minimize RNAse and proteinase activity, such as by including RNAse inhibitors and protease inhibitors. For instance, an S100 extract of a microbe may be prepared by ultracentrifugation of a whole cell extract at 100,000×g, followed by fractionation on an anion exchange column, for instance, a Q-sepharose column. Retained polypeptides may be removed by a cation gradient, such as NaCl. Fractions with the polypeptides of interest may be identified by determining which fractions have psiRNAs (Hale et al., 2008, RNA 14, 2572-2579). Those fractions with psiRNAs may be further separated on a second anion exchange column, eluted with a cation gradient, and the fractions with psiRNAs identified again. Those fractions with psiRNAs may be further separated on a cation exchange column, for instance, an S-sepharose column, and bound polypeptides eluted with a cation gradient. The complex of polypeptides bound to a psiRNA may be identified by native gel Northern analysis (Hale et al., 2008, RNA 14, 2572-2579).

Optionally, the complex of polypeptides bound to a psiRNA identified by native gel Northern analysis can be subjected to tandem mass spectroscopy. Tandem mass spectroscopy analysis is routinely used by the skilled person to identify polypeptides. The data from the analysis can be used to search readily available databases containing genomic sequence data for the microbe used as a source of the polypeptides, and the coding sequences encoding the polypeptides can be easily identified. These coding sequences can be easily obtained by, for instance, PCR amplification of genomic DNA, and inserted into vectors. The cloned coding regions encoding polypeptides useful in the methods described herein may be expressed in a microbe (for use in in vivo methods described herein), and optionally obtained from the microbe for use in in vitro methods described herein. Polypeptides present in the peak fractions from the cation exchange column may also be used in in vitro methods.

The present invention also includes genetically modified microbes. A genetically modified microbe may have a polynucleotide encoding a psiRNA, a polynucleotide encoding a Cmr1, a Cmr2, a Cmr3, a Cmr4, a Cmr5, or a Cmr6 polypeptide, or a combination thereof. Compared to a control microbe that is not genetically modified according to the present invention, a genetically modified microbe may exhibit production of an exogenous polynucleotide or an exogenous polypeptide disclosed herein or a fragment thereof, or increased production of an endogenous polypeptide disclosed herein. A polynucleotide encoding a psiRNA or a polypeptide disclosed herein may be present in the microbe as a vector or integrated into a chromosome. Examples of microbes that can be genetically modified include, but are not limited to, eukaryotic cells, such as S. cerevisiae and P. pastoris, bacteria, such as gram-negative microbes, for example, E. coli, and archaea, such as Haloferax volcanii.

Also provided herein are methods for inactivating a polynucleotide. The methods include incubating under suitable conditions a composition that includes a target polynucleotide, a psiRNA, and polypeptides that will catalyze the inactivation, for instance cleavage of the specific target polynucleotide. The methods of the present invention may occur in vitro or in vivo. In some aspects of the in vivo methods, the polypeptides that will catalyze the inactivation of the target polynucleotide are endogenous to the microbe in which the in vivo method is occurring. Restriction endonucleases recognize a specific nucleotide sequence of a target polynucleotide and cleave the target at a specific location. For instance, EcoRI recognizes a target double stranded DNA at GAATTC, and cleaves the double stranded molecule at a specific and predictable location within that recognition sequence. Cleaving a target polynucleotide using the methods of the present invention permits a level of flexibility that is not available with restriction endonucleases having a specific recognition site. A target polynucleotides described herein are not limited to those possessing a specific recognition site. Moreover, using the methods presented herein, the skilled worker can determine where cleavage of a target polynucleotide is desired, and then design a psiRNA that will guide the polypeptides described herein to cleave the target at that specific location. Moreover, unlike restriction endonucleases known in the art, the target polynucleotide may be RNA.

Current evidence indicates that the psi-tag is a universal feature of the psiRNAs that function as guides for the various effector complexes of the CRISPR-Cas system in diverse prokaryotes (Marraffini et al., 2008, Science, 322:1843-1845, and Brouns et al., 2008, Science, 321:960-964). The psi-tag is found on the psiRNAs in both Staphylococcus epidermidis and E. coli, where evidence indicates that silencing occurs at the DNA level (Marraffini et al., 2008, Science, 322:1843-1845, and Brouns et al., 2008, Science, 321:960-964). The methods disclosed herein for inactivating a target polynucleotide include the use of polynucleotides having a 5′ region that is present irrespective of the target polynucleotide. This is in contrast with the structure of silencing RNAs known to function in eukaryotic cells, which do not possess conserved sequence elements.

As disclosed above, a psiRNA has a 5′ region and a 3′ region. The 5′ region of a psiRNA may be selected from a repeat disclosed herein, or may be selected as described herein from other repeats present in a microbe containing CRISPR loci. psiRNAs function with polypeptides encoded by a subset of coding regions typically physically located near CRISPR loci (referred to as cas genes) to result in inactivation of a target polynucleotide. For example, psiRNAs function with the Cmr proteins to effect cleavage of target RNA polynucleotides (Example 2). Thus, when the method includes the use of a 5′ region present in a particular microbe, the method may further include the use of Cmr polypeptides from that microbe. In view of the present disclosure, the skilled person now knows which psiRNAs can be used with polypeptides having endonuclease activity, for instance, endoribonuclease activity, to result in inactivation of a target polynucleotide.

In those aspects where the method is in vitro, Cas polypeptides derived from P. furiosus such as those described herein may be used with a psiRNA having a psiRNA-tag that is identical to or having sequence similarity with, for instance, GAAUUGAAAG (SEQ ID NO:19), AAUUGAAAG, AUUGAAAG, UUGAAAG, UGAAAG, or GAAAG, or another psiRNA-tag present in P. furiosus. Alternatively, polypeptides useful in the methods may be obtained from another microbe having CRISPR loci as described herein, and these polypeptides may be used with a psiRNA having a psiRNA-tag present in that same microbe. Conditions suitable for inactivation of a target polynucleotide for example by the Cmr polypeptides described herein may include incubation for an hour at a suitable temperature such as at least 30° C., at least 40° C., at least 50° C., at least 60° C., at least 70° C., at least 80° C., and at least 90° C. Other conditions suitable for inactivation include a buffer, for instance, 20 mM HEPES, an appropriate pH, such as 7.0, and additional components such as KCl (250 mM), MgC12 (1.5 mM), ATP (1 mM), DTT (10 mM), and an RNAse inhibitor.

In vivo methods may be used to decrease or eliminate expression of polypeptides encoded by a target polynucleotide, or decrease or eliminate activities associated with non-coding RNAs. For instance, a psiRNA may be introduced into a microbe to inactivate a target polynucleotide. Such methods may be used to decrease or eliminate expression of an endogenous polynucleotide or an exogenous polynucleotide. Such methods may be used to immunize a microbe against an exogenous polynucleotide, for instance, a bacteriophage, conjugative plasmid, or transposon. The psiRNA may be introduced as an RNA polynucleotide, or may be introduced as a vector encoding the psiRNA. The vector may be one that is unable to replicate in the microbe, able to replicate, or integrate into the microbe's genome.

In those aspects where the method is in vivo, and the microbe includes polypeptides that catalyze the inactivation of a target polynucleotide (e.g. cleavage of a polynucleotide target by the Cmr complex as described herein), the psiRNA used in the method typically includes a psiRNA-tag that is present in that microbe. For instance, when the microbe is P. furiosus or Sulfolobus solfataricus, the psiRNA-tag may be GAAUUGAAAG (SEQ ID NO:19), AAUUGAAAG, AUUGAAAG, UUGAAAG, UGAAAG, or GAAAG, or another psiRNA-tag present in P. furiosus or S. solfataricus. When the method is in vivo, and the microbe does not include polypeptides that catalyze the inactivation of a target polynucleotide, the microbe may be engineered to express the polypeptides. For instance, polynucleotides encoding Cmr polypeptides may be expressed in a cell, and a psiRNA including a psiRNA-tag present in P. furiosus may be used. Alternatively, polypeptides from some other microbe having CRISPR loci and Cmr (or other Cas) polypeptides may be used as the source of polynucleotides encoding polypeptides having the inactivating, for instance, endoribonuclease activity. The polynucleotides may be introduced into and expressed in a microbe, and the psiRNA used in such a microbe would likewise be obtained from the same microbe that was the source of the Cas polypeptides.

Examples of microbes that may be used in methods of the present invention include, but are not limited to, microbes useful in starter cultures, probiotic cultures, dietary supplement cultures, and cultures for use in fermentation of biomass for production of biofuels. Examples of such microbes include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Erwinia spp., Yersinia spp., Bacillus spp., Vibrio spp., Legionella spp., Pseudomonas spp., Neisseria spp., Bordetella spp., Helicobacter spp., Listeria spp., Agrobacterium spp., Staphylococcus spp., Streptococcus spp., Enterococcus spp., Clostridium spp., Corynebacterium spp., Mycobacterium spp., Treponema spp., Borrelia spp., Francisella spp., Brucella spp., Bifidobacterium spp., Brevibacterium spp., Propionibacterium spp., Lactococcus spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp., and Oenococcus spp.

With respect to the 3′ region, typically, the nucleotide sequence of the target is known. Since the 3′ region of a psiRNA used in a method disclosed herein is complementary or substantially complementary to the target polypeptide that is to be inactivated by the psiRNA, the sequence of the target polynucleotide will dictate the nucleotide sequence of a psiRNA 3′ region.

The methods of the present invention are not limited to any target polynucleotide. A target polynucleotide may be an endogenous polynucleotide sequence (such as a DNA or a microbial mRNA) or an exogenous polynucleotide sequence. The genomic sequence of many bacterial and archaeal microbes are published, thus the skilled person can easily select a series of nucleotides from a genomic sequence that can be used as a target polynucleotide. In embodiments that use a target polynucleotide that is a portion of an mRNA or a non-coding RNA, computer algorithms for identifying genomic sequences encoding such RNAs, for example open reading frames, are routinely used in the art. Moreover, as the speed of acquiring microbial genomic sequences continues to increase, more microbial genomes become readily available.

In some embodiments a target polynucleotide is ribonucleotide. Examples of ribonucleotides include, but are not limited to, ribonucleotides encoding a polypeptide (e.g., mRNAs) and non-coding ribonucleotides (e.g., small non-coding RNAs, or ncRNAs). Examples of ncRNAs include, but are not limited to, ribosomal RNA, bacterial signal recognition particle RNA, transfer RNA, transfer-messenger RNA, small nuclear RNA, small nucleolar RNA, and ribonuclease P. The target polynucleotide may be the deoxynucleotide sequence that encodes such ribonucleotides.

Examples of ribonucleotides encoding a polypeptide include, but are not limited to, mRNAs encoding secreted polypeptides, polypeptides associated with the outer membrane (such as porins and receptors), polypeptides associated with the inner membrane, periplasmic polypeptides, mitochondrial polypeptides, structural polypeptides (such as fimbriae, flagella, polypeptides involved in protein export), polypeptides involved in biosynthesis (such as biosynthesis of amino acids, polypeptides, nucleotides, DNA, RNA, lipids, sugar residues, coenzymes, prosthetic groups, non-ribosomal polypeptides), polypeptides involved in metabolism (such as the phosphotransferase system for glucose and other sugars, glycolysis, the pentose phosphate pathway, the Entner-Doudoroff pathway, the tricarboxylic acid cycle, pathways for polyols, pathways for carboxylates), polypeptides involved in energy production (pathways for electrons to oxygen, pathways for anaerobic electron transport), DNA restriction and modification polypeptides, polypeptides involved in protein degradation, polypeptides involved in motility and chemotaxis, polypeptides involved in resistance to antibiotics (such as beta-lactamases), and ATP-coupled solute transport polypeptides. The target polynucleotide may be the deoxynucleotide sequence that encodes such ribonucleotids.

Other examples of ribonucleotides include RNA sequences encoded by microbial invaders such as bacteriophage able to infect a microbe that contains a CRISPR locus, conjugative plasmids, microbial transposons, and the like. The target polynucleotide may be the deoxynucleotide sequence of the bacteriophage, conjugative plasmid, or transposon. Examples of bacteriophage include, but are not limited to, members of virus families Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, and Tectiviridae.

Examples of psiRNAs that may be used include the sequence 5′-N₅₋₁₀NNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNN(SEQ ID NO:1) or 5′-N₅₋₁₀NNNNNNNNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNNNNNNNN (SEQ ID NO:2), where N₅₋₁₀ is the 5′ region of between 5 and 10 nucleotides and the last 31 nucleotides of SEQ ID NO:1 and the last 37 nucleotides of SEQ ID NO:2 are the guide sequence and are complementary or substantially complementary to the target. The psiRNA is designed to not only be complementary to the target polynucleotide, but in some cases, also to line up the desired cleavage site on the target polynucleotide with the arrow when the psiRNA and the target polynucleotide are hybridized. Cleavage of the target polynucleotide by the Cmr complex occurs, for example, opposite the position defined by the arrow. Thus, knowledge of the nucleotide sequence of a target polynucleotide surrounding a desired cleavage site aids in the design of the appropriate psiRNA.

The present invention also provides kits. A kit may include one or more of the polynucleotides or polypeptides described herein. For instance, a kit may include a psiRNA or a vector encoding a psiRNA, or a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, a Cmr5 polypeptide, a Cmr6 polypeptide, or a combination thereof. In another aspect, a kit may include one or more vectors encoding one or more of the polypeptides described herein. Kits may be used, for instance, for modifying a microbe to express polypeptides described herein, or for in vitro cleaving of a target polynucleotide. The kit components are present in a suitable packaging material in an amount sufficient for at least one assay. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. Instructions for use of the packaged polypeptide or primer pair are also typically included.

As used herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the invention nucleic acids can be used for methods as described herein. In addition, the packaging material contains instructions indicating how the materials within the kit are employed. As used herein, the term “package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits a kit component. Thus, for example, a package can be a glass vial used to contain milligram quantities of a polypeptide or polynucleotide. “Instructions for use” typically include a tangible expression describing the reagent concentration or at least one assay method parameter.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example 1

In many prokaryotes, non-coding RNAs that arise from the CRISPR loci are now thought to mediate defense against viruses and other molecular invaders by an RNAi-like pathway. CRISPR (clustered regularly interspaced short palindromic repeat) loci contain multiple short regions of similarity to invader sequences separated by short repeat sequences, and are associated with resistance to infection by corresponding viruses. It is hypothesized that RNAs derived from these regions, termed prokaryotic silencing (psi)RNAs, guide Slicer-like complexes of partner proteins to destroy invader nucleic acids. Here we have investigated CRISPR-derived RNAs in the archaeon Pyrococcus furiosus. Northern analysis revealed multiple RNA species consistent with a proposed biogenesis pathway that includes full-length CRISPR locus transcripts and intermediates generated by endonucleolytic cleavages within the repeat sequences. However, our results identify the principal products of the CRISPR loci as small psiRNAs comprised primarily of invader-targeting sequence with perhaps only 5-10 nucleotides of CRISPR repeat sequence. These RNAs are the most abundant CRISPR RNA species in P. furiosus and are likely the guides for the effector complexes of the proposed prokaryotic RNAi (pRNAi) system. We analyzed cell-free extracts fractionated under non-denaturing conditions and found that the various CRISPR RNA species are components of distinct RNA-protein complexes, including at least two complexes that contain mature-length psiRNAs. Finally, RNAs are produced from all 7 CRISPR loci present in the P. furiosus genome and interestingly, the most recently acquired psiRNAs encoded proximal to the leader sequence of a CRISPR locus appear to be the most abundant.

Methods and Materials

Small RNA cloning and sequencing. Total RNA was isolated from P. furiosus cells using the Trizol reagent (Invitrogen) as indicated by the manufacturer. Approximately 300 μg of total RNA was separated on an 8×8.5 cm 15% polyacrylamide 7M urea gel using DNA size standards (pGEM markers, Promega) for size determination. RNAs between 20 and 50 nucleotides were isolated and passively eluted overnight in 0.5 M ammonium acetate, 0.1% SDS, 0.5 mM EDTA, followed by ethanol precipitation. In order to remove potential 5′ triphosphates or cap structures, RNAs were treated with 50 U tobacco acid pyrophosphatase (TAP) (Epicentre) for 2 hours at 37° C. The eluted RNAs were cloned using standard microRNA cloning protocols (Lau et al., 2001, Science, 294:858-862) using the following primers containing EcoRI restriction sites: 3′ adapter: 5′-AppTTTAACCGCGAATTCCAGddC-3′ (IDT) (SEQ ID NO: 31), 5′ adapter: 5′-ACGGAATTCCTCACTrArArA-3′ (IDT) (SEQ ID NO:32), RT/PCR primer: 5′-GACTAGCTGGAATTCGCGGTTAAA-3′ (IDT) (SEQ ID NO:33), PCR primer: 5′-CAGCCAACGGAATTCCTCACTAAA-3′ (SEQ ID NO:34). An additional PCR was performed in order to add a BanI restriction site (GGYRCC) for concatamerization of the PCR products using the following primers: PCR2 5′ primer 5′-GACTAGCTTGGTGCCGAATTCGCGGTTAAA-3′ (SEQ ID NO:35), PCR2 3′ primer 5′-GAGCCAACAGGCACCGAATTCCTCACTAAA-3′ (SEQ ID NO: 36). The products were subject to restriction digestion and DNA ligation by standard methods (Lau et al., 2001, Science, 294:858-862). cDNAs were cloned into the pCRII TOPO vector (Invitrogen) and transformed into TOP10 cells (Invitrogen) as described by the manufacturer. Plasmid preparation and sequencing was performed in a 96-well plate format using standard M13 forward, reverse, and T7 promoter primers. Sequences were analyzed using BLAST (NCBI). Northern analysis. Approximately 10 μg of P. furiosus total RNA was separated on 15% polyacrylamide 7 M urea gels (Criterion, Bio-Rad) alongside [32P]-5′-end radiolabeled RNA markers (Decade, Ambion). The RNAs were transferred onto nylon membranes (Zeta-Probe, Bio-Rad) using a Trans-Blot SD Semi-Dry Cell (Bio-Rad). Membranes were baked at 80° C. for at least an hour before pre-hybridization in a ProBlot hybrdization oven (LabNet) for at least 1 hour at 42° C. Pre-hybridization and hybridization was performed in either Oligo-UltraHyb (Ambion) buffer or hybridization buffer containing 5×SSC, 7% SDS, 20 mM sodium phosphate, pH 7.0 and 1×Denhardt's solution. Deoxyribonucleotide probes (MWG) (20 pmol) were 5′ end labeled with T4 Polynucleotide Kinase (Ambion) and γ-[32P]-ATP (specific activity >7,000 Ci/mmol, MP Biomedicals) using standard protocols. Labeled probes were added to the pre-hybridization buffer, followed by hybridization overnight at 42° C. Following hybridization, two washes were performed in 2×SSC, 0.5% SDS for 30 minutes at 42° C. Resulting blots were exposed to a phosphoimager screen for 24-72 hours and scanned. The probes and sequences that were used are given in Table 2.

TABLE 2 Probes. Probe Sequence (SEQ ID NO:) repeat 1, 5,  CTTTCAATTCTATTTT(AG)GTCTTATTC(GT)AAC 6 (SEQ ID NO: 37) (antisense) repeat 1, 5,  GTT(AC)CAATAAGAC(TC)AAAATAGAATTGAAAG 6 (SEQ ID NO: 38) (sense) repeat 2, 4,  CTTTCAATTCTTTTGTAGTCTTATTGGAAC 7 (SEQ ID NO: 39) (antisense) repeat 2, 4,  GTTCCAATAAGACTACAAAAGAATTGAAAG 7 (SEQ ID NO: 40) (sense) psiRNA 1.01 GGTCAGATCAGATTGCTTAAGACAAGAAATG (antisense) (SEQ ID NO: 41) psiRNA 1.01 CATTTCTTGTCTTAAGCAATCTGATCTGACC (sense) (SEQ ID NO: 42) psiRNA 2.01 GTGGAGCAGAGTCAGAAGAAGAAGTGCG (antisense) (SEQ ID NO: 43) psiRNA 2.01 CGCACTTCTTCTTCTGACTCTGCTCCAC (sense) (SEQ ID NO: 44) psiRNA 4, 02 TCTGATAGGCTTCAAAGAGTGGCGCTTCAAC (antisense) (SEQ ID NO: 45) psiRNA 4.02 GTTGAAGCGCCACTCTTTGAAGCCTATCAGA (sense) (SEQ ID NO: 46) psiRNA 5.02 GGGAATGGTTCACGTAGTACTTGAGGGCGC (antisense) (SEQ ID NO: 47) psiRNA 5.02 GCGCCCTCAAGTACTACGTGAACCATTCCC (sense) (SEQ ID NO: 48) psiRNA 6.01 CTAAGGACATTTGTACGTCAAATTCTTCAC (antisense) (SEQ ID NO: 49) psiRNA 6.01 GTGAAGAATTTGACGTACAAATGTCCTTAG (sense) (SEQ ID NO: 50) psiRNA 7.01 GCTCTCAGCCGCAAGGACCGCATAC (antisense) (SEQ ID NO: 51) psiRNA 7.01 GTATGCGGTCCTTGCGGCTGAGAGC (sense) (SEQ ID NO: 52) psiRNA 7.11 CCTTATATGGGTGTTGTGAAGCAGGATAGAAC (antisense) (SEQ ID NO: 53) psiRNA 7.11 GTTCTATCCTGCTTCACAACACCCATATAAGG (sense) (SEQ ID NO: 54) psiRNA 7.21 GGCTCTACCTAATCATCCTCTTGACACAAC (antisense) (SEQ ID NO: 55) psiRNA 7.21 GTTGTGTCAAGAGGATGATTAGGTAGAGCC (sense) (SEQ ID NO: 56) psiRNA 8.01 GACTGTGTGTGGAGCAGCTATTTGCTTCGGC (antisense) (SEQ ID NO: 57) psiRNA 8.01 GCCGAAGCAAATAGCTGCTCCACACACAGTC (sense) (SEQ ID NO: 58)

Chromatography. 15 g of Pyrococcus furiosus cells were lysed anerobically in 200 mL 50 mM Tris, pH 8.0 in the presence of 4 mg/L RNase-free DNase (Sigma). The extract was subject to ultracentrifugation at 113,000× g for 2 hours (Optima L-90K, Beckman-Coulter). The resulting S100 extract was applied to a 60 mL DEAE Sepharose-FF column and eluted using a 0-500 mM NaCl gradient. The resulting fractions were analyzed by isolating RNAs from 250 μl of each 30 mL fraction using the Trizol LS protocol (Invitrogen). The RNAs were separated on 10% polyacrylamide 7 M urea gels, blotted, and subject to Northern analysis as described above, using the Oligo-UltraHyb hybridization buffer for both pre-hybridization and hybridization. See above for probe sequences.

Native Northern analysis. 40 ul of DEAE fractions from peaks A-C were separated on a 4-20% polyacrylamide gel (Bio-Rad) using SDS-free running buffer (25 mM Tris, 19.2 mM glycine). De-proteininzed samples were analyzed in parallel. Gels were run at 50 V for 3-4 hours at room temperature. The gel was soaked in 5M urea, 45 mM Tris, 45 mM boric acid, 1 mM EDTA for 15 minutes, then subject to blotting and Northern analysis as described above, using Ultra-Hyb Oligo hybridization buffer (Ambion) and a probe against psiRNA 7.01.

Results

psiRNAs cloned from the seven CRISPR loci in Pyrococcus furiosus The P. furiosus genome contains seven CRISPR loci, each encoding between 11 and 51, and together encoding 200 potential psiRNAs (Grissa et al., 2007, Bioinformatics, 8:172) (FIG. 1). To investigate whether psiRNAs are produced from the 7 CRISPR loci, we isolated and cloned small RNAs (less than 50 nucleotides) from total P. furiosus RNA preparations. Among 872 small RNA clones sequenced, 144 (17.3%) were derived from CRISPR loci. In addition, 42.2% corresponded to rRNA, 23.9% were derived from ORFs, and 12.4% were from sRNAs (snoRNA homologs). The remaining 4.2% of sequences were derived from tRNAs, transposons, Hhc RNAs (Klein et al., 2002, Proc Natl Acad Sci USA, 99:7542-7547) and intergenic sequences.

Most of the CRISPR clones consisted primarily of psiRNA (variable) sequence and included some flanking repeat sequence. The clones included 64 of the 200 potential P. furiosus psiRNAs and represented all seven CRISPR loci (FIG. 1). We have adopted a simple system of nomenclature for psiRNAs, in which the psiRNA is designated by a 3-digit number. The first digit indicates the locus number (1, 2 and 3-8 in P. furiosus) and the second two digits, separated from the first by a decimal point, indicate the position of the psiRNA within that locus (relative to the leader). For example, the first psiRNA in CRISPR locus 1 is Pf psiRNA 1.01, the first psiRNA in CRISPR locus 2 is psiRNA 2.01, and the last psiRNA in CRISPR locus 1 is psiRNA 1.51 (see FIG. 1). FIG. 1 shows the number of times each individual psiRNA was cloned. The psiRNA clones ranged between 17 and 50 nucleotides in length (see Table 3 for psiRNA clone sequences). The clones included variable amounts of the psiRNA sequence (12 to 40 nucleotides) and of repeat sequence at the 5′ (0 to 8 nucleotides) and/or 3′ (0 to 22 nucleotides) end. In addition to the psiRNA clones, we isolated a few CRISPR-derived clones that lacked psiRNA sequence and consisted of a portion of leader sequence upstream of a repeat (See “Leader” section of Table 3), indicating that the 3′ end of the CRISPR leader is also transcribed.

TABLE 3 Cloned psiRNA sequences SEQ ID Sequence NO: psiRNA Locus 1 CTGATCTGACCAGAGCTGGTTCCAATAAGACTAAA  59 1.01 CTGATCTGACCAGAGCTGGTTCCAAGTAAGACTAA  60 1.01 CTGATCTGACCAGAGCTGGTTCCAATAAGACTAAA  61 1.01 CAATCTGATCTGACCAGAGCTGGTTCCAAT  62 1.01 CAATCTGATCTGACCAGAGCTGGTTCCAAT  63 1.01 ATGATTCATTTCTTGTCTTAAGCAAT  64 1.01 TTGTCTTAAGCAATCTGATCT  65 1.01 CACTAAAGTCATACTTTACTGCTACAACCCGCTCTGG  66 1.04 GTCATACTTTACTGCTACAACCCGCTCTGG  67 1.04 TACTGCTACAACCCGCTCTGGGTCGAG  68 1.04 TTACTGCTACAACCCGCTCTGGGTCGA  69 1.04 ACTGCTACAACCCGCTCTGGGTTGA  70 1.04 CTGACACGAACATAAACAGTTCCAATAAGACTACAGAAGA  71 1.06 CTGACACGAACATAAACAGTTCCAATAAGACTACAGAAGA  72 1.06 GAAAGGGAAATGTGCGTAAAGGTTTTCTTCCC  73 1.07 GAAAGGGAAATGTGCGTAAAGGTTTTCTTCCC  74 1.07 TTGACCCACCACCAGCCCTGTTCCAATAAGAC  75 1.08 GAAAGGCGTGCCGTGTGTTTTTATAA  76 1.11 GAAAGGCGTGCCGTGTGTTTTTATAA  77 1.11 GTTGCTGCATATCCAGTGTGG  78 1.12 GTTGCTGCATATCCAGTGTGG  79 1.12 GGCAAGTTCTGGCCTATACTGTCTCCTAATGTCT  80 1.13 TAGGAGACAGTATAGGCCAGAACTTGCCCAG  81 1.13 GACATTAGCNGACAGTATAGGCCA  82 1.13 TTAGCAAATTGCCGATTACTGCACATAAAAAAAATAG  83 1.14 CTATAAGGGATTGAAAGGTCAAAGGTATAN  84 1.16 CTATAANGGATTGAAAGGTCAAGGGTATACT  85 1.16 TTCGCGGTTAAACAATCTGATCTGACCAGAGCTGGTTCCAAT  86 1.19 GGACAGCGTGGACACGGTGAACGGGCTCTGGA  87 1.21 CTGATAGAACCTTTGCCACC  88 1.24 CTGATAGAACCTTTGCCACC  89 1.24 CATACTTGCGGATACGGATCCAGTCAAAACTTGACTG  90 1.26 TTGCGGATATGGATCCAGTCAAAACTTGACTG  91 1.26 GCGGATACGGATCCAGTCAAAACTTGACTG  92 1.26 GAAAGCATACTTGCGGATACGGATCCAGT  93 1.26 CTCTGGGTCGTCTATGTTTTTGA  94 1.27 GAGTAGAAATGCCCAAATTCCCCTTAGGGACA  95 1.36 TTTGTGATAGTGTTCTTTGCAACGAAGTGCTTGCTGGTCAG  96 1.43 GTTTGTGATAGTGTTCTTTGCAACGAAGAGCTTGCTGG  97 1.43 GAGTGCCCCGAGCCGGGGGCT  98 1.49 Locus 2 CTGACACGAACATAAACAGTTCCAATAAGACTACAGAAGA  99 2.02 CTGACACGAACATAAACAGTTCCAATAAGACTACAGAAGA 100 2.02 ATGGCTCGATGGAATTATGTTCCAATAAGACTACAAAAG 101 2.03 ATGGCTCGATGGAATTATGTTCCAATAAGACTACAAAAG 102 2.03 CTAACTAACATCACCAATAATTAATTGTAAGTTAG 103 2.10 GCTACCATGGCCATCACCAATAATTAATTGTAAGT 104 2.10 CTGAGCCAACCCACCACTTTGGTAAAACT 105 2.13 CTGAGCCAACCCACCACTTTGGTAAAACT 106 2.13 CTGAGCCAACCCACCACTTTGGTAAAACT 107 2.13 TGAGGCTGGAGAGGGCTTCTTTGTTACTACTTGCGT 108 2.17 TTATGTTCATGTTCCACATCTAA 109 2.18 TATGTTCATGTTCCACACTA 110 2.18 Locus 4 TTGAAAGGAATGTTGCTCAATGCAAAGGGCTCACCGCTGCTGGTGTTCCA 111 4.01 CTCAATGCAAAGGGCTCACCGCTGCTGGTGTTCCAATAAGA 112 4.01 CTCACCGCTGCTGGTGTTCCAATAAGACTACAAAAGA 113 4.01 TTGAAAGTTGAGTTGAAGCGCCACTCTTTGAA 114 4.02 TTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGAG 115 4.02 ATTGAAAGTTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGA 116 4.02 AGTTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGAGT 117 4.02 GTTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGAGT 118 4.02 TTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGAGTT 119 4.02 AAGTCGGGTCCCTTGGAGTTCCGAACGGGCTCCCGAGGCTGTTCCA 120 4.04 GGGCTCCCGAGGCTGTTCCAATAAGAC 121 4.04 GTTGATTCCCTTATAGATGTTCGTTTTCCACA 122 4.05 ATGTTCGTTCTCGTTCACTGTTATTCTCTT 123 4.07 CTCGTTCACTGTTATTCTCTTT 124 4.07 AAAACTAAAAAAAGAAGAGGTGGTGGTGAAGAAT 125 4.08 GAAAGTCTCAATTGGGGAGTTGCTTTAATGGCTTTT 126 4.12 TCAATCCGAGAATCGAATTTTCCTATACGCTTTTGTT 127 4.21 TTTGTTTTTGCTCCTGTGTCTTGTGGTGATAAAATG 128 4.22 TTTGTTTTTGCTCCTGTGTCTTGTGGTGATAAAATG 129 4.22 GTGATAAAATGTTACAATAAGACTACAAAAG 130 4.22 Locus 5 ATTGAAAGGACCATACTCACCAGCAGCGGTGAGCCCTTTGCATTGA 131 5.01 ATTGAAAGGACCATACTCACCAGCAG 132 5.01 GTTCACGTAGTACTTGAGGGCGCTCACGTTACAATAAGACCA 133 5.02 TTTCANGCAGTACTTGAGGGCGCTCATGTTNCANTANGACCAA 134 5.02 AAGAAGGGGAATGGTTCACGTAGCTACTTGAGGGC 135 5.02 CAATAATACAGTCCTAATGCTCGTG 136 5.03 CAATAATACAGTCCTAATGCTCGTG 137 5.03 TTGAAAACGCTAGCAGGACTAGTGCTTGT 138 5.04 CGCTAGCAGGACTAGTGCTTGTG 139 5.04 CTTCTCGAATCTATCGAATTCGGTTACAATAAGACCAAAATAGA 140 5.09 AGCCACATAANACATTGTCATACAAAGTATGACAAAATA 141 5.11 CACATAAGACATTGTCATACAAAGTAGGACAAAA 142 5.11 AAGACATTGTCATACAAAGTAGGACAAA 143 5.11 GTCCTCTTGGAGACCGTTCCTGTTACAATAAGACCA 144 5.12 GTCACGTAATTCGCCAAGTCCNCNT 145 5.12 AATAGTTACAATAAGACCAAAATA 146 5.15 CTAGCTTTTCACACACTCT 147 5.18 TAAACTANGNTGATTTTGTAAT 148 5.20 GAAAGAGTATTCCACCGAGAATTGTGCC 149 5.29 GTATTCCACCGAGAATTGTGCCTTTGTACTGGACTG 150 5.29 Locus 6 TCTATTTTAGTCTTATTGTAACGTTCCACTAAGGAC 151 6.01 TTTAGTCTTATTGTAACGTTCCACTAAGGAC 152 6.01 AATTTGACGTACAAATGTCCTTAGTGGAAC 153 6.01 TTCGGGACCTGTAGGTCGTTACAATAAGACTAAAATAGA 154 6.02 GTTAATGGTAAAGTTACAATAAGACTAAA 155 6.03 GTTCTGCCGTCCCTTTTCTCGACG 156 6.09 TTCTGCCGTCCCTTTTCTCGACGAACCTCATACCGA 157 6.09 TTCTGCCGTCCCTTTTCTCGACGAAC 158 6.09 TTCTGCCGTCCCTTTTCTCGACGAAC 159 6.09 TATAGGCGGAACTCCCT 160 6.13 AAGTGTTTTCGAATATTGTTACTTCTTGTGT 161 6.15 CTATAAGACTGAAACTTCACACCT 162 6.37 TTAACACTCTTAACCCCAG 163 6.38 GTCCAAAAACGTTACAATAAGACTAAA 164 6.39 TTAAGCTGGGATGGGCTATATACAAAGACAG 165 6.42 AATTCTGGAAGGTTGTAGAAA 166 6.44 Locus 7 CGCCCACCTTTGTTACGTTCCAATAAGACT 167 7.01 TTGTAGTATGCGGTCCTTGCGGCTGAGAGCACTTCAG 168 7.01 TTGTAGTATGCGGTCCTTGCGGCTGAGAGCA 169 7.01 TTGTAGTATGCGGTCCTTGCGGCTGAGAGCA 170 7.01 GTAGTATGCGGTCCTTGCGGCTGAGAGCA 171 7.01 GAAAGTTGTAGTATGCGGTCCTTGC 172 7.01 GTCTTCGATTAGTGAAAACAGTTCCAATAAGACTACAAAAG 173 7.02 GTCGTTATCTCTTACGAAGTCTTCGATTAGT 174 7.02 GTTACACGTGAGTGCAAGNTCCAATAAGACTACAAAAGA 175 7.04 GTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 176 7.04 GTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 177 7.04 GTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 178 7.04 TTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 179 7.04 TTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 180 7.04 TTTACACGTGAGTGCAAGTTCCAATAAGACTACAAAAGA 181 7.04 ACAAAAGAATTGAAAGTTAACCTCCTT 182 7.07 AGTTATCTAAGCTCTGCTTAAATGGGAAAATCTTATAAG 183 7.14 Locus 8 GAAGAGGAAGAAATGCAGACGACGTGATAAACTACGTGAA 184 8.02 CAGACGACGTGATAAACTACGTGAAAAGTT 185 8.02 AACTTTTCAACGTAGTTTATCACGTCGTCTGA 186 8.02 GTGCACTAAGGCACCATACGCCCAA 187 8.03 ATTGAAGCTTGCCCAACCTCTCTAGAAACGCCCA 188 8.06 AATTGAAGNNAAAATCTCTTTTTAAATCTTTGA 189 8.07 ATTGAAGCGCANATCTNTTTTTAAATCTTTGA 190 8.07 Leaders GTAGGAGTATTGGGGCAAAAAAGCCCCCTGTTCCAATAAGAC 191 before 2 or 7 GGGGGGAATTGGGGCAAAAAAGCCCCCTGTTCCAATAAGACT 192 before 2 or 7 GGGGGAATTGGGGCAAAAAAGCCCCCTGTTCCAATAAGACTAC 193 before 2 or 7 GGGGGAATTGGGGCAAAAAAGCCCCCTGTTCCAATAAGACTAC 194 before 2 or 7 CCCCCTGTTCCAATAAGACTACAAAAG 195 before 2 or 7 CCCCCTGTTCCAATAAGACTACAAAAG 196 before 2 or 7 AAGCCCCCTGTTCCAATAAGACTACAAA 197 before 2 or 7 GAAAAAGCCCCCTGTTACAATAAGACCAA 198 before 5 GAAAAAGCCCCCTGTTACAATAAGACCAAAATAGA 199 before 5 TTAGGAGTATTGGGGCGAAAAAGCCCCCTGTTACAATAAGACTA 200 before 6 GGGGGAATTAGGGCAAAAAAGCCCACTGTTCCAATAAGACT 201 before 8 GGGGAATTAGGGCAAAAAAGCCCACTGTTCCAATA 202 before 8 Repeat sequences are indicated in bold.

Our sampling is not apparently at saturation, however, we cloned psiRNAs from the beginning, middle and end of CRISPR loci (FIG. 1), indicating that RNAs are produced from across the length of the loci. Interestingly, however, the likelihood of cloning was significantly higher for psiRNAs encoded within the first part of a CRISPR locus, suggesting a greater abundance in the organism of psiRNAs from these regions. Two-thirds of the psiRNAs that we cloned were from the first third of their CRISPR locus and 45% were one of the first four psiRNAs in a given locus (FIG. 1). With the exception of locus 2, this trend was observed within each individual CRISPR locus.

Comparison of the percentage of psiRNAs cloned from a given locus (Table 4, % of clones) to the percentage of the total psiRNAs encoded by that locus (Table 4, % of psiRNAs) revealed that most of the loci are represented proportionately within the clones. However, locus 6 seems to be significantly underrepresented in the cDNA library. Locus 6 encodes ˜22% of the psiRNAs in P. furiosus, however only ˜12% of the cloned RNAs were derived from this locus. This suggests that the psiRNAs encoded within locus 6 are less abundant in P. furiosus than those encoded by the six other CRISPR loci.

TABLE 4 Distribution of cloned psiRNAs. CRISPR # of # of % of % of locus psiRNAs Clones psiRNAs Clones 1 51 40 25% 30% 2 20 12 10%  9% 4 22 20 11% 15% 5 30 20 15% 15% 6 45 16 22% 12% 7 21 17 10% 13% 8 11 7  5%  5% Total 200 132

The results of the RNA cloning suggest the presence of novel, small psiRNAs in P. furiosus. However, the cloned RNAs were not of a uniform size or composition. To determine whether discrete psiRNA species are present in P. furiosus, we undertook additional analysis.

Northern analysis of RNAs derived from the CRISPR loci in P. furiosus In order to further investigate the RNAs that arise from the CRISPR loci in P. furiosus, we undertook Northern analysis with probes against both repeat and psiRNA sequences. Probes were designed for detection of both sense (transcription from the leader sequence) and antisense RNAs. FIG. 2A shows results obtained with a probe that recognizes the repeat sequence (sense orientation) that is common to P. furiosus CRISPR loci 1, 5 and 6. This probe detected a prominent band at ˜65 nucleotides, a less prominent band at ˜130 nucleotides, and an unresolved set of bands of greater than 150 nucleotides near the top of the gel. For this and all other probes tested, no significant differences in the patterns were observed from total RNA samples prepared with and without DNase treatment indicating that the bands represent RNAs. Consistent with the observations and CRISPR RNA processing pathway proposed by others (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72; Markova et al., 2006, Biol Direct, 1:7; Sorek et al., 2008, Nat Rev Microbiol, 6:181-186), the set of bands above 150 nucleotides likely represents a mixture of primary transcripts from the 3 loci as well as larger intermediates generated by cleavages within repeat regions. The most prominent band detected with the repeat probe in P. furiosus (˜65 nucleotides) corresponds well to the primary product of the CRISPR loci reported previously in other organisms (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72), however in this work this RNA is identified as the “1× intermediate” (FIG. 2). This band corresponds in length to a psiRNA (˜35-40 nts) and repeat (˜30 nts), and likely represents psiRNAs with flanking repeat sequences generated by cleavages within the adjacent repeats (see FIG. 2C). The detection of this RNA by the repeat probe suggests that cleavage may be asymmetric within the repeat sequence, leaving a substantial contiguous region of the 30 nucleotide repeat on one side (e.g. the 3′ end as modeled in FIG. 2C) for efficient detection by Northern probes. A less abundant band of ˜130 nucleotides corresponds in length to two psiRNAs with flanking repeat sequences and likely represents the immediately upstream 2× intermediates (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72) (FIG. 2C).

Northern analysis with a probe against one of the variable psiRNA sequences revealed novel CRISPR RNA species. Using a probe against psiRNA 4.02 (sense orientation), we detected a band at ˜60 nucleotides and a very faint signal near the top of the gel, but the most prominent band is ˜46 nucleotides (FIG. 2B) and corresponds in size to that of the psiRNA (35 nts in the case of psiRNA 4.02) and ˜10 nucleotides of repeat sequence. A significant secondary band was detected at ˜39 nucleotides (FIG. 2B). Importantly, similar results were observed in Northern analysis of RNAs from other CRISPR loci. Results for all RNAs analyzed, both sense and antisense, are compiled in FIG. 3. First, like the repeat 1, 5, 6 probe, a probe for the repeat sequence common to loci 2, 4 and 7 (sense orientation) detected prominent diffuse bands of ˜65 and ˜130 nucleotides (theoretical 1× and 2× processing intermediates, see FIG. 2C). In addition, we probed for psiRNAs from the first part of each CRISPR locus (1.01, 2.01, 4.02, 5.02, 6.01, 7.01 and 8.01) as well as for psiRNA sequences from the middle and end of locus 7 (7.11 and 7.21). Strikingly, probes for each of the psiRNA sequences (sense orientation) detected a single predominant RNA species (indicated with dots in FIG. 3). Most of these predominant RNAs were ˜43 to ˜46 nucleotides. The observed size of the major RNA species was generally 5 to 10 nucleotides longer than the encoded psiRNA sequence. The psiRNA with the longest observed primary product (psiRNA 1.01) has an unusually long psiRNA sequence. These findings, together with the observation that these RNAs are recognized by psiRNA but not repeat sequence probes, suggest that the primary psiRNA species in P. furiosus consists of a psiRNA with ˜5-10 nucleotides of repeat.

In addition to the primary psiRNA species, each of the psiRNA probes detected other RNAs. These often included an RNA close to the size of the 18 nucleotide 1× intermediates that were detected by repeat probes, and in some cases (e.g. psiRNAs 101 and 402) an RNA was detected that was the size of the theoretical 2× intermediate. Many of the psiRNA probes detected other faint bands. However, in many cases the most prevalent secondary species was a slightly smaller RNA of ˜38 to ˜45 nucleotides.

We did not detect antisense RNAs with most of the CRISPR probes (FIG. 3B). Prominent bands were detected with probes from psiRNAs 2.01 and 7.11, however the absence of corresponding bands with the repeat probes suggests that these are not CRISPR locus-derived RNAs.

Northern analysis of CRISPR RNA distribution in fractionated P. furiosus extract The CRISPR RNAs are hypothesized to function in complex with proteins in various aspects of RNA-guided genome defense in prokaryotes (Markova et al., 2006, Biol Direct, 1:7). To assess whether the CRISPR-derived RNA species that we identified may be components of distinct complexes, we analyzed the distribution of the RNAs across fractions from anion exchange chromatography of P. furiosus S100 cell extract performed under anaerobic conditions. Fractions were evaluated by Northern analysis using probes against the repeat sequence common to loci 1, 5 and 6, and psiRNA 4.02 and 7.01 sequences (FIG. 4). For reference, the profile of RNAs detected in unfractionated extract is shown in the first lane.

The distribution of the various RNAs across the fractions suggests the presence of several distinct CRISPR RNA-containing complexes. The novel primary and secondary psiRNAs (˜45 and ˜39 nts) from both loci co-fractionated in a distinct set of fractions denoted as peak A (FIG. 4, fractions 10-14). Other larger CRISPR RNA species were not observed in peak A. The primary psiRNA is also present in peak B (fractions 20-23) along with a fraction of the 1× intermediate RNA and some of the variable-size psiRNA species. However, the highest concentration of the 1× intermediate is found in a distinct set of fractions that lack small psiRNAs, termed peak C (fractions 23-26). The mixture of RNAs that likely includes full CRISPR locus transcripts and larger intermediates, and the 2× intermediate are found primarily in peak D (fractions 31-34). These results suggest the presence of multiple complexes each containing distinct subsets of CRISPR-derived RNAs in P. furiosus.

To verify the presence of CRISPR RNA-protein complexes in the fractionated P. furiosus extract, we examined the complexes on native gels. Using the probe for psiRNA 7.01, we compared the mobility of the RNAs, both in the presence of the co-fractionating proteins in peaks A, B and C and following protein extraction, by non-denaturing PAGE and Northern analysis. In each case, a significant shift in the mobility of the RNAs was observed in the presence of the proteins. Peak D was not examined on native gels. Together our results indicate that the novel primary psiRNA is a component of at least 2 distinct RNA-protein complexes (peaks A and B), the 1× intermediate is found primarily in a third complex (peak C), and a fourth complex includes larger psiRNA precursors. These complexes are likely candidates for the mediators of psiRNA production, invader destruction and CRISPR element integration in the proposed prokaryotic RNAi pathway.

Discussion

Novel CRISPR RNAs. The CRISPR loci found in many prokaryotes encode alternating repeat and “spacer” or psiRNA sequences, and have been shown to give rise to a series of RNAs that decrease in increments from the full-length locus transcript to a single psiRNA and repeat (i.e. 1× intermediate, see FIG. 2C) (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72). Current evidence indicates that processing occurs by endonucleolytic cleavages within the repeat sequences (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481). The RNA products of the CRISPR loci are hypothesized to guide silencing of viruses and other genome invaders. In this work, we have identified a novel class of smaller discrete CRISPR-derived RNAs that we have termed psiRNAs, which appear to be the ultimate gene products of the CRISPR loci (FIG. 2C).

The primary psiRNA species that we have identified is the most abundant CRISPR-derived RNA detected in P. furiosus. The primary psiRNAs are approximately 5 to 10 nucleotides longer than the corresponding psiRNA sequence (i.e. approximately 45 nucleotides long). These RNAs are shorter than the smallest discrete CRISPR RNA products previously reported (i.e. the ˜60-65 nt 1× intermediate species) (Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481; Lillestol et al., 2006, Archaea, 2:59-72), and are presumably generated by exonucleolytic processing of the 1× intermediate. Our Northern and sequence analysis indicates that these RNAs are comprised primarily of psiRNA sequence and do not contain substantial repeat sequence. A secondary psiRNA of about 39 nucleotides was also consistently observed among psiRNA profiles.

CRISPR RNA-protein complexes. The common primary and secondary psiRNA species are likely candidates for the guide RNA component of the effector complex in the proposed pRNAi genome defense pathway. Both of these psiRNA species, but not larger intermediate CRISPR RNAs, are found in RNA-protein complexes in anion exchange chromatography peak A (FIGS. 4 A and B), thus peak A could contain the effector complex. The primary psiRNA is even more abundant in peak B, which contains relatively less of the secondary psiRNA but also contains some 1× intermediate RNA (FIG. 4). Peak B may also contain the effector complex and/or a complex involved in the exonucleolytic processing of the 1× intermediate to psiRNAs. The 1× intermediate RNA is most abundant in peak C, which is adjacent to peak B and processing may occur across peak B and C fractions. Longer CRISPR RNAs are found in peak D. Our results indicate that the various RNA species are components of distinct RNA-protein complexes in P. furiosus. Extensive purification and analysis will determine whether these hypothesized activities and the Cas proteins predicted to function in CRISPR RNA biogenesis and invader silencing (e.g. RNA binding proteins and nucleases) are present in these complexes.

psiRNA expression. The psiRNAs are hypothesized to act in a manner similar to the antibodies of the human immune system and expression would be expected even in the absence of active infection to patrol for returning invaders. Our results indicate that psiRNAs are actively produced from all 7 CRISPR loci in P. furiosus. Moreover, expression levels appear to be equivalent between the loci under the growth conditions examined with the possible exception of one locus that yielded 50% fewer psiRNA clones than expected (see locus 6, Table 4). Our results confirm that CRISPR RNAs are transcribed from the leader sequence in P. furiosus and indicate that a portion of the leader sequence is also transcribed.

Interestingly, we found evidence of significantly higher levels of expression of psiRNAs encoded proximal to the leader of a CRISPR locus. Current data indicate that these are the most recently acquired psiRNA sequences within CRISPR loci (Pourcel et al., 2005, Microbiology, 151:653-663; Barrangou et al., 2007, Science 315:1709-1712). Increased levels of these psiRNAs may be important for targeting current invaders. Distal psiRNAs may be produced at lower levels to provide surveillance for past invaders. It is not clear whether the increased level of leaderproximal psiRNAs results from differences in RNA transcription (e.g. partial transcription of the loci), processing, stability or other factors. This is an important new aspect of understanding the regulation of psiRNA expression that remains to be explored.

Example 2

Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. The system arises from clustered regularly interspaced short palindromic repeats (CRISPRs) that harbor short invader-derived sequences, and CRISPR-associated (Cas) protein-coding genes. Here we have identified an apparent CRISPR-Cas effector complex that employs small CRISPR RNAs (termed prokaryotic silencing or psiRNAs) to recognize and destroy corresponding target RNAs. The complex consists of psiRNAs and a subset of Cas proteins termed the RAMP module (or Cmr) proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3′ end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5′ sequence tag but have distinct 3′ ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs recognized by the psiRNAs.

Experimental Procedures

Chromatography: P. furiosus S100 extract was prepared from approximately 4 grams of cells. Cells were resuspended in 20 mL of 50 mM Tris (pH 7.0), 100 U RNase-free DNase (Promega), and 0.5 mM phenylmethanesulphonyl fluoride (PMSF) at room temperature by stirring. The resulting whole cell extract was subject to ultracentrifugation at 100,000×g for 1.5 hours using an SW 41 Ti rotor (Beckman). The resulting S100 extract was loaded onto a 5 mL Q-sepharose Fast Flow (GE) pre-packed column. Proteins were eluted using a0-1 M NaCl gradient. Fractions were analyzed by Northern analysis by isolating RNA from 100 ul of each fraction using Trizol LS (Invitrogen, following manufacturer's instructions). The RNAs were separated on 15% TBE-urea gels (Criterion, Bio-Rad), blotted and analyzed for the presence of a single guide sequence as described previously (Hale et al., 2008, RNA, 14:2572-2579). Peak fractions containing the psiRNA doublet were further separated on a second 5 mL Q-sepharose column, eluted with 220-430 mM NaCl. Fractions were analyzed as described above. Peak fractions were pooled, diluted in 50 mM sodium phosphate buffer, pH 7.0, and loaded onto a 5 mL S sepharose column (GE). Bound proteins were eluted with a gradient of 0-1 M NaCl. Native gel northern analysis was performed as described previously (Hale et al., 2008, RNA, 14:2572-2579). The secondary data shown in Table 5 was obtained from S100 extract fractionated on a DEAE column as previously described (Hale et al., 2008, RNA, 14:2572-2579) followed by a hydroxyapatite column eluted with a gradient of 5-500 mM sodium phosphate buffer, pH 6.5, and further purified by native gel electrophoresis.

TABLE 5 Proteins identified by tandem mass spectrometry of native RNA-protein complexes. All proteins that were identified in the S-native sample (FIG. 5), and in a native band from a separate chromatography scheme, HA-native. Numbers represent the % coverage, with the number of unique peptides in parentheses. Protein S-native HA-native Annotated Function Cas proteins PF1129 56.0 (54)  26.9 (20) hypothetical protein PF1129 PF1128 40.4 (13) 20.5 (5) hypothetical protein PF1128 PF1126 58.3 (14) 17.6 (3) hypothetical protein PF1126 PF1124 16.2 (5)  11.8 (4) hypothetical protein PF1124 PF0352 28.1 (5)  hypothetical protein PF0352 PF1125 24.9 (5)  hypothetical protein PF1125 PF1130 3.6 (1) hypothetical protein PF1130 Non-Cas proteins PF1717 76.2 (28) translation initiation factor IF-2 gamma subunit PF1683 73.6 (19) N-acetyl-gamma-glutamyl-phosphate reductase PF0990 60.6 (26) phenylalanyl-tRNA synthetase beta subunit PF1685 59.0 (20) acetylornithine/acetyl-lysine aminotransferase PF0481 55.7 (7)  translation initiation factor IF-2 beta subunit PF1827 53.8 (14) 20.8 (4) hypothetical protein PF1827 PF1881 51.6 (4)  chromatin protein PF0989 45.1 (22) phenylalanyl-tRNA synthetase alpha subunit PF0124 34.3 (14) hypothetical protein PF0124 PF1140 30.2 (7)  translation initiation factor IF-2 alpha subunit PF0495 29.7 (34) reverse gyrase PF1204 29.2 (11) seryl-tRNA synthetase PF1264 26.1 (3)  translation initiation factor IF-5A PF0351 25.6 (8)  hypothetical protein PF0351 PF1238 23.9 (14) putative ABC transporter PF1615 23.1 (18) hypothetical protein PF1615 PF0496 21.2 (5)  hypothetical protein PF0496 PF0594 18.4 (4)  14.3 (2) ornithine carbamoyltransferase PF1405 16.6 (10) 12.9 (7) cleavage and polyadenylation specifity factor protein PF0547 15.8 (5)  hypothetical protein PF0547 PF0969 14.7 (4)  2-ketovalerate ferredoxin oxidoreductase subunit alpha PF0220 14.1 (6)  13.6 (7) hexulose-6-phosphate synthase PF1375 13.3 (6)  elongation factor Tu PF1976 12.1 (6)  L-aspartate oxidase PF0666 11.5 (6)  nol1-nop2-sun family putative nucleolar protein IV PF1746 11.0 (6)  hypothetical protein PF1746 PF0251 11.0 (4)  hypothetical protein PF0251 PF1579 10.4 (7)  DNA topoisomerase VI subunit B PF0966 10.4 (4)  2-oxoglutarate ferredoxin oxidoreductase PF0533 10.1 (7)  indolepyruvate ferredoxin oxidoreductase subunit a PF1578 9.2 (3) DNA topoisomerase VI subunit A PF0026 8.8 (4) tRNA nucleotidyltransferase PF1540 8.7 (5) ADP forming acetyl coenzyme A synthetase PF1203 8.1 (5) formaldehyde:ferredoxin oxidoreductase PF1046 7.9 (3) queuine trna-ribosyltransferase PF0464 7.5 (4) glyceraldehyde-3-phosphate:ferredoxin oxidoreductase PF1768 5.1 (2) 2-oxoglutarate ferredoxin oxidoreductase PF0440 3.9 (5) ribonucleotide-diphosphate reductase alpha subunit PF1843 1.7 (2)  7.3 (6) chromosome segregation protein smc PF0102  76.6 (15) hypothetical protein PF0102 PF1883  74.9 (13) small heat shock protein PF1548  63.3 (24) hypothetical protein PF1548 PF1931 27.9 (6) hypothetical protein PF1931 PF0162 11.2 (2) hypothetical protein PF0162 PF0204  6.0 (2) hypothetical protein PF0204 PF1871  3.7 (1) “N(2),N(2)-dimethylguanosine tRNA methyltransferase” PF1245  2.2 (1) hypothetical d-nopaline dehydrogenase PF1167  1.5 (1) chromosome segregation protein

Protein assignment by tandem mass spectrometry: In-gel and in-solution tryptic digests were performed as previously described (Lim et al., 2008, J Proteome Res, 7:1251-1263; Wells et al., 2002, Mol Cell Proteomics, 1:791-804). Desalted tryptic peptides were analyzed by nLC-MS/MS on a linear ion-trap (LTQ, ThermoFisher) as previously described (Lim et al., 2008, J Proteome Res, 7:1251-1263). Acquired data was searched against a P. furiosus-specific database (forward and inverted) using the TurboSEQUEST algorithm (ThermoFisher). Data was collated and filtered to obtain a 1% false discovery rate at the protein level using the ProteoIQsoftware package (BioInquire) that is based on the PROVALT algorithm (Weatherly et al., 2005, Mol Cell Proteomics, 4:762-772).

Cloning and sequencing of psiRNAs from the purified complexes: RNAs from S-column fractions (isolated as described above for Northern analysis) were treated with 1 U calf intestinal alkaline phosphatase (Promega) for 1 hour at 37° C., followed by extraction with phenol:chloroform:isoamyl alcohol (PCI; pH 5.2, Fisher) and ethanol precipitation. The resulting RNAs were separated by 15% polyacrylamide, TBEureagels (Criterion, Bio-Rad), visualized by SYBR Gold staining (Invitrogen) and the visible bands were excised. RNAs were passively eluted overnight in 0.5 M ammonium acetate, 0.1% SDS, 0.5 mM EDTA, followed by ethanol precipitation. A 5′-phosphorylated, 3′ capped oligonucleotide (5′-pCTCGAGATCTGGATCCGGG-ddC3′; IDT (SEQ ID NO:26) was ligated with T4 RNA ligase to the 3′ end of the RNAs. The ligated RNAs were PCI extracted, ethanol precipitated, gel purified, and subject to reverse transcription using Superscript III (Invitrogen) RT (as described by the manufacturer), followed by gel purification. The gel-purified cDNAs were polyA-tailed for 15 minutes at 37° C. using terminal deoxynucleotide transferase (Roche) using manufacturer's recommendations. PCR was performed to amplify the cDNA libraries using the following primers: 5′-CCCGGATCCAGATCTCGAG-3′ (SEQ ID NO:27), 5′-GCGAATTCTGCAG(T)30-3′ (SEQ ID NO:28)). cDNAs were cloned into the TOPO pCRII (Invitrogen) cloning vector and transformed into TOP10 cells. White and light-blue colonies were chosen for plasmid DNA preparation, and sequencing using the M13 Reverse and T7 promoter sequencing primers was performed by the University of Georgia Sequencing and Synthesis Facility.

Small RNA deep sequencing: Small RNA libraries were prepared using the Illumina small RNA Sample preparation kit as described by the manufacturer (Illumina). Briefly, total RNA was isolated from P. furiosus and fractionated on a 15% polyacrylamide/urea gel, and small RNAs 18-65 nt in length were excised from the gel. 5′ and 3′ adapters were sequentially ligated to the small RNAs and the ligation products were gel-purified between each step. The RNAs were then reverse-transcribed and PCR-amplified for 16 cycles. The library was purified with a Qiagen QuickPrep column and quantitated using an Agilent Bioanalyzer and a nanodrop. The sample was diluted to a concentration of 2 μM and subjected to 42 cycles of sequencing on the Illumina Genome Analyzer II.

Small RNA Analysis: Sequence data was extracted from the images generated by the Illumina GenomeAnalyzer II using the software applications Firecrest and Bustard. The adapter sequences were then trimmed from the small RNA reads, which were then mapped to the P. furiosus genome using btbatchblast. Only reads that mapped perfectly to the genome over their entire length were used for further analysis. The location and number of reads that initiate within the CRISPR repeats were determined using a perlscript. As the maximal read length of the sequences was 42 nt, it was not possible to be certain that the 3′ end of a read represented the actual 3′ end of the small RNA. Therefore, the deep sequencing data was only used to determine the most frequent 5′ ends and the number of reads that map to each psiRNA.

Nuclease assays: To detect target RNA cleavage, 2 μL of the peak S-column fractions (FIG. 5C) or 500 nM each of recombinant proteins was incubated with 0.05 pmoles of 32P-5′ end-labeled synthetic target RNAs (FIGS. 3, 4 and 5) and 0.5 pmoles of each unlabeled psiRNA (FIG. 9) for 1 hour at 70° C. in 20 mM HEPES pH 7.0, 250 mM KCl, 1.5 mM MgCl2, 1 mM ATP, 10 mM DTT, in the presence of 1 unit of SUPERase-In ribonuclease inhibitor (Applied Biosystems). For assay with recombinant proteins, the psiRNAs were first incubated with the proteins for 30 minutes at 70° C. prior to the addition of target RNA. Reaction products were isolated by treatment with 800 ng of proteinase K for 30 minutes at room temperature, followed by PCI extraction and ethanol precipitation. The resulting RNAs were separated by 15% polyacrylamide, TBE 7M urea gels and visualized by phosphorimaging. 5′ end-labeled RNA size standards (Decade Markers, Applied Biosystems) were used to determine the sizes of the observed products. Annealed RNAs were prepared by mixing equimolar amounts of RNAs in 30 mMHEPES pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate and incubating for 1 minute at 95° C., followed by 1 hour at 37° C. Annealing was confirmed by nondenaturing 8% PAGE.

Expression and purification of recombinant proteins: The genes encoding P. furiosus Cmr1-1 (PF1130), Cmr2 (PF1129), Cmr3 (PF1128), Cmr4 (PF1126), Cmr5 (PF1125) and Cmr6 (PF1124) were amplified by PCR from genomic DNA or existing constructs and cloned into a modified version of pET24d (PF1124, PF1125 and PF1126) or pET200D (PF1128, PF1129 and PF1130). The recombinant proteins were expressed in E. coli BL21-RIPL cells (DE3, Stratagene). The cells (400 mL cultures) were grown to a OD600 of 0.7, and expression of the proteins was induced with 1 mM isopropyl-″-D-thiogalactopyranoside (IPTG) overnight at room temperature. The cells were pelleted, resuspended in 20 mM sodium phosphate buffer (pH 7.6), 500 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and disrupted by sonication. The sonicated sample was centrifuged at 4,500 rpm for 15 min at 4° C. The supernatant was heated at 75-78° C. for 20 min, centrifuged at 4,500 rpm for 20 min at 4° C., and filtered (0.8 μm pore size Millex filter unit, Millipore). The recombinant histidine-tagged proteins were purified by batch purification using 50 μl Ni-NTA agarose beads (Qiagen) equilibrated with resuspension buffer. Following 3 washes (resuspension buffer), the bound proteins were eluted with resuspension buffer containing 500 mM imidazole. The protein samples were dialyzed at room temperature against 40 mM HEPES (pH 7.0) and 500 mM KCl prior to performing activity assays.

Synthetic psiRNAs The 45- and 39-nucleotide psiRNAs were chemically synthesized (Integrated DNA Technologies). The sequence of the 45-nucleotide psiRNA 7.01 is: ATTGAAAGTTGTAGTATGCGGTCCTTGCGGCTGAGAGCACTTCAG (SEQ ID NO:29). The sequence of the 39-nucleotide psiRNA 7.01 is:

(SEQ ID NO: 30) ATTGAAAGTTGTAGTATGCGGTCCTTGCGGCTGAGAGCA.

Results

Isolation of a complex containing mature psiRNAs and a subset of Cas proteins. PsiRNAs are hypothesized to guide Cas proteins to effect invader silencing in prokaryotes (Browns et al., 2008, Science, 321:960-964; Hale et al., 2008, RNA, 14:2572-2579; Makarova et al., 2006, Biol Direct, 1:7). P. furiosus is a hyperthermophilic archaeon whose genome encodes 200 potential psiRNAs (organized in seven CRISPR loci) and at least 29 potential Cas proteins (largely found in 2 gene clusters), including 5 core Cas proteins and 3 sets of additional Cas proteins: the Cmr, Cst and Csa proteins (see FIG. 5F). In P. furiosus, most psiRNAs are processed into 2 species of ˜45 nucleotides and ˜39 nucleotides (Hale et al., 2008, RNA, 14:2572-2579). To gain insight into the functional components of the CRISPR-Cas invader defense pathway, we isolated complexes containing both of the mature psiRNA species from P. furiosus cellular extract through a series of steps of non-denaturing chromatography on the basis of psiRNA fractionation profiles (FIG. 5). The doublet of psiRNAs, detectable both by Northern blotting of an individual psiRNA and total RNA staining (SYBR), was purified away from larger CRISPR-derived RNAs (including the 1× intermediate; Hale et al., 2008, RNA, 14:2572-2579) as well as other cellular RNAs (FIG. 5C). To determine whether the psiRNAs are components of RNA-protein complexes in the purified fraction (FIG. 5C), we performed native gel northern analysis. The mobility of the psiRNAs on native gel electrophoresis was reduced in the purified fraction relative to a sample from which proteins were extracted (FIG. 5D), indicating the presence of psiRNA-protein complexes in the purified fraction.

To determine whether Cas proteins may be components of the psiRNP identified by native northern analysis (FIG. 5D), we gel purified the psiRNA-containing complex from the native gel and analyzed the sample by mass spectrometry. The sample contained a mixture of proteins that included seven Cas proteins identified with 99% confidence: Cmr1-1, Cmr1-2, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6 (FIG. 5E).

The identities of the non-Cas proteins found in the sample are listed in Table 5. Analysis of a native gel-purified psiRNP obtained by an alternate chromatography scheme revealed a similar Cas protein profile (Cmr2, Cmr3, Cmr4, and Cmr6), but few common non-Cas proteins (Table 5). The five common co-purifying non-Cas proteins are denoted in Supplemental Table 5. None of these proteins has any known link to the CRISPR-Cas system.

Remarkably, the seven Cas proteins associated with the complex are all encoded by the tightly linked cmr genes (Haft et al., 2005, PLoS Comput Biol, 1:e60). Moreover, the identified proteins comprise the complete set of Cmr proteins (Haft et al., 2005, PLoS Comput Biol, 1:e60). (The independently defined “polymerase cassette” is closely related to the RAMP module (Makarova et al., 2006, Biol Direct, 1:7).) There are 6 cmr genes: cmr2 encodes a predicted polymerase with HD nuclease domains, and cmr1, cmr3, cmr4, and cmr6 encode repeat-associated mysterious proteins (RAMPS) (Haft et al., 2005, PLoS Comput Biol, 1:e60; Makarova et al., 2002, Nucleic Acids Res, 30:482-496). The P. furiosus genome contains two cmr1 genes and a single representative of each cmr2-cmr6, and all seven corresponding proteins were found in the purified psiRNP complex (FIG. 5E). The organization of the genes encoding the seven identified proteins is shown in FIG. 5F. Six of the seven identified Cas proteins are encoded in a nearly contiguous region of one of the two major cas gene loci in P. furiosus. This locus is located directly adjacent to CRISPR locus 7, and also encodes core Cas proteins Cas1-Cas4 as well as Cas6. The striking correlation between the evolutionary co-segregation and physical association of the 6 Cmr proteins strongly supports the co-function of the proteins. Our findings indicate that the two mature psiRNA species are components of complexes containing the RAMP module or Cmr proteins in P. furiosus.

psiRNAs possess a 5′ psiRNA-tag sequence. In order to better understand the nature of the two psiRNA species that are components of the purified complexes, each of the two RNA bands present in the final chromatography sample (FIG. 6A) was extracted and cloned. We obtained sequences of 53 RNAs (20 from the upper band and 31 from the lower band) that included psiRNAs from all seven P. furiosus CRISPR loci (Table 6). Six RNAs with the same guide sequence were represented in both the upper and lower bands, consistent with Northern analysis that has shown that most psiRNAs exist in both size forms (Hale et al., 2008).

TABLE 6 Cloned psiRNAs. Sequences for all of the clones obtained from both the upper and lower band of the S-column material (See FIG. 7). The length and the origin of each psiRNA is shown. For the sequences, repeat se- quence is shown in bold, and the sequences are aligned by the repeat sequences. SEQ ID  psiRNA sequence length psiRNA NO. Upper Band  ATTGAAAGTTAGCAAATTGCCGATTATTGCACATAAAAAAAATAG 45 1.14 203  ATTGAAAGTTAGCAAATTGCCGATTATTGCACATAAAAAAAATAG 45 1.14 204  ATTGAAAGACTGGATTGAGAGCAACTTGTCGAATTATGTCGTCAA 45 1.40 205 AATTGAAAGTGTTCATCAGCACTTCTTCTTCTGACTCTGCTCC 43 2.01 206 AATTGAAAGTGTTCATCGCACTTCTTCTTCTGACTCTGCTCC 42 2.01 207  ATTGAAAGCTAATTTACGCTTTAGCTCGTGATCAACCCTAATC 43 2.19 208  ATTGAAAGCTAATTTACGCTTTAGCTCGTGATCAACCC 38 2.19 209  ATTGAAAGTTGAGTTGAAGCGCCACTCTTTGAAGCCTATCAGAGT 45 4.02 210  ATTGAAAGGCTTCAGGTCTTCAATATTCAATCCCGGTCCCTTTCA 45 4.03 211  ATTGAAAGGCTTCAGGTCTTCAATATTCAATCCCGGTCCCTTTCA 45 4.03 212     GAAAGTCTCTACCCTTACAAGCTTCTCGAATCTATCGAATTC 42 5.09 213     GAAAGGTCACGTAATTCGCCAAGTTCTCTTGGATACCGTTC 41 5.12 214  ATTGAAAGGTGGATAATATAATCCCTGTTTTTCCCAAGA 39 5.13 215  ATTGAAAGTGGAACTCTATCAAGGTTTGCAACACCTTGCTCCCGC 45 5.24 216     GAAAGGACAAAGAACTCCCTAGCGTCCCTCCCCGTGTA 38 6.07 217  ATTGAAAGTGGGGTCTCGTCGCAATCGGTGCAGTATTCCTAAGCC 45 6.26 218  ATTGAAAGATCTCCATCATACCAATGCTGTGCAAAATCAATCTTG 45 6.40 219  ATTGAAAGTAAACTTAAGCTGGGATGGGCTATATACAAAGACAGA 45 6.42 220 AATTGAAAGTCAAGAGTTCTATCCTGCTTCACAACACCCATATAA 46 7.11 221  ATTGAAAGGCGTTAATGAACAATAAGCCTGACACGAACATAAA 43 1.06, 2.02 222 Bottom Band  ATTGAAAGCCGGTTCTGCACCCGAAACTTTCATACCAAA 39 1.03 223 AATTGAAAGCCGGTTCTGCACCCGAAACTTTCATACCAA 39 1.03 224  ATTGAAAGGTAGTGAGGCGTTGAACTTGACCCACCACCA 39 1.08 225  ATTGAAAGTGAGTTGTTTAGTCTAACTCTTACACCATC 38 1.19 226  ATTGAAAGTGCGCTATTCTCGGGTCAAGCCTCCCAGCCT 39 1.22 227     GAAAGCACCACCACGATGAAGGTACCGTTTTCAAC 35 1.37 228     GAAAGCACCACCACGATGAAGGTACCGTTTTCAAC 35 1.37 229  ATTGAAAGTGTTCATCGCACTTCTTCTTCTGAC 33 2.01 230  ATTGAAAGCTTCTTCGAAGTCGTAGTTTAGTGTGTCAAG 39 2.05 231  ATTGAAAGTTCTAGAAGTTCTCTTGCGAGAGCCAGGAGC 39 2.06 232     GAAAGCTAATTTATGCTTTAGCTCGTGATCAACCCTA 37 2.19 233     GAAAGCTAATTTACGCTTTAGCTCGTGATCAACC 34 2.19 234        AGGAATGTTGCTCAATGCAAAGGGCTCACCGCT 33 4.01 235      AAAGTCTCAATTGGGGAGTGCTTTAATGGCTTTT 34 4.12 236  ATTGAAAGGGAACTCCTCGATTTTAGTACCTGTGTC 36 5.05 237  ATTGAAAGCCACATAAGACATTGTCATACAAAGTAGG 37 5.11 238  ATTGAAAGGTCACGTAATTCGCCAAGTCCTCTTGGAGA 38 5.12 239  ATTGAAAGGTGGATAATATAATCCCTGTTTTTCCCAAGA 39 5.13 240  ATTGAAAGGTGGATAATATAATCCCTGTTTTTCCCAAGA 39 5.13 241  ATTGAAAGGTGGATAATATAATCCCTGTTTTTCCCAAGA 39 5.13 242  ATTGAAAGGTGGATAATATAATCCCTGTTTTTCCCAAGA 39 5.13 243  ATTGAAAGCAGTTCTACTTTGATAAGACTGTGGTGGTTA 39 6.03 244  ATTGAAAGGACAAAGAACTCCCTAGCGTCCCTCCCCGTG 39 6.07 245 AATTGAAAGTTCTGCCGTCCCTTTCTCGACGAACCTCAT 39 6.09 246  ATTGAAAGGCACCTTCTTCACCATCGCCGTCTGGATTGC 39 6.14 247        AGTTGTAGGCTCGTGGACTTGGCTTCCACACAACTA 36 6.24 248  ATTGAAAGTATCTATTGTACAGGTACTTGTTACACGT 37 7.14 249  ATTGAA GCTTGCCCAACCTCTCTAGAAACGCCCAC 35 8.06 250  ATTGAAAGGCGTTAATGAACAATAAGCCTGACACGAAC 38 1.06, 2.02 251  ATTGAAAGTAATCTCAATAACTTTGGCTTCTTTTCTGTG 39 4.14, 5.19 252  ATTGAAAGTAATCTCAATAACTTTGGCTTCTTTTCTGTG 39 4.14, 5.19 253  ATTGAAAGACACGAATCCCCAACATTCTTCACCACCCCT 39 NF 254  ATTGAAAGTGACTGCCTCCCTCAGAACCTTAATGAT 36 NF 255

The cloned psiRNAs consisted primarily of an individual guide (invader-targeting or “spacer”) sequence, however, all of the clones retained a portion of the common repeat sequence at the 5′ end. Indeed, the majority (˜70%) of the RNAs in both bands contained an identical 5′ end consisting of an 8-nucleotide segment of the repeat sequence (FIG. 6A). The difference between the two psiRNA size forms was found at the 3′ ends. Downstream of the repeat sequence, the majority of the clones from the top band contained 37 nucleotides of guide sequence (the full length of a typical guide element in P. furiosus) (FIG. 6A, top panel). The 3′ ends of most of the clones from the bottom band were located within the guide sequence. The majority of these RNAs contained 31 nucleotides of guide sequence downstream of the repeat sequence (FIG. 6A, bottom panel).

The psiRNAs are processed from long CRISPR locus transcripts (Brouns et al., 2008, Science, 321:960-964; Hale et al., 2008, RNA, 14:2572-2579; Lillestol et al., 2006, Archaea, 2:59-72; Lillestol et al., 2009, Mol Microbiol, 72:259-272; Tang et al., 2002, Proc Natl Acad Sci USA, 99:7536-7541; Tang et al., 2005, Mol Microbiol, 55:469-481) (FIG. 6B). In P. furiosus, the Cas6 endoribonuclease cleaves CRISPR RNAs at a site within the repeat element located 8 nucleotides upstream of the guide sequence, generating the precise 5′ end observed in the two psiRNA species found in the complex (FIG. 6B; (Carte et al., 2008, Genes Dev, 22:3489-3496)). Our results indicate that the 5′ end generated by the Cas6 endoribonuclease is maintained in the mature psiRNAs, but that the RNAs undergo further processing at the 3′ end to generate psiRNAs that contain either ˜37 or ˜31 nucleotides of guide sequence (FIG. 6B). The mechanism that defines the two distinct 3′ end boundaries is not known. The larger ˜45-nucleotide mature psiRNA species is generally more abundant than the smaller 18-nucleotide species (Hale et al., 2008, RNA, 14:2572-2579, FIGS. 1 and 2A).

The short repeat sequence that remains at the 5′ end of mature psiRNAs P. furiosus provides a common identifying sequence tag for the psiRNAs that could function in recognition of the RNAs by the proteins in the CRISPR-Cas pathway. In order to rigorously delineate the potentially important psiRNA-tag or “psi-tag”, we purified small RNAs from P. furiosus, performed deep sequencing and obtained the sequences of the 5′ ends of more than 10,000 CRISPR-derived RNAs (from loci 1-7). The 5′ ends of the majority of the RNAs mapped 8 nucleotides upstream of the guide sequence (FIG. 6C), verifying the presence of a discrete psi-tag on small CRISPR-derived RNAs in P. furiosus. Analysis of RNAs isolated from Solfolobus solfataricus and Sulfolobus tokodaii revealed that 8-nucleotide psi-tags (repeat-derived sequence at the 5′ end of the psiRNAs, as indicated) are also present in the psiRNAs of these species (FIG. 11). The Pyrococcus and Sulfolobus species are members of the extremely phylogenetically diverse Euryarchaeta and Crenarchaeota kingdoms, respectively. These results and similar data from psiRNAs of the bacteria, E. coli (Brouns et al., 2008, Science 321, 960-964.) and S. epidermidis (Marraffini et al., 2008, Science, 322:1843-1845) support the highly conserved nature and functional importance of the psi-tag.

The sequences of CRISPR repeats (from which psi-tags are derived) are generally conserved within groups of organisms, but can vary widely (Godde and Bickerton, 2006, J Mol Evol, 62:718-729; Kunin et al, 2007, Genome Biol, 8:R61). Thus, while the sequence of the psi-tag found on most P. furiosus psiRNAs (AUUGAAAG) can be found in the repeat sequence of numerous organisms, psi-tags of distinct sequence and length would be expected in others. We found evidence to support this prediction in the psiRNAs from P. furiosus CRISPR locus 8, which contains a single nucleotide deletion in the psi-tag region of the repeat. Of the 640 RNAs obtained by deep sequencing that mapped to CRISPR locus 8, 60% had a 7-nucleotide AUUGAAG psi-tag. In E. coli, CRISPR transcripts are cleaved by a different endoribonuclease (Cse3 of the Cse complex), which nonetheless appears to generate RNAs with an 8-nucleotide AUAAACCG repeat sequence at the 5′ end (Brouns et al., 2008, Science, 321:960-964), suggesting that this is a general feature of the psiRNAs. Interestingly, the distinct CRISPR repeat sequences found in various genomes are accompanied by specific subsets of Cas proteins (Kunin et al, 2007, Genome Biol, 8:R61), which may reflect coupling of specific series of Cas proteins with the psi-tagged RNAs that they recognize.

Homology-dependent cleavage of a target RNA. One hypothesis for the mechanism by which CRISPR RNAs and Cas proteins mediate genome defense is psiRNA-guided cleavage of invader RNAs, analogous to Slicer function in eukaryotic RNAi pathways (Farazi et al., 2008, Development, 135:1201-1214; Girard and Hannon, 2008, Trends Cell Biol, 18:136-148; Hutvagner and Simard, 2008, Nat Rev Mol Cell Biol, 9:22-32; Makarova et al., 2006, Biol Direct, 1:7). Therefore, we tested the ability of the isolated psiRNP complexes to cleave a 5′ end-labeled target RNA complementary to endogenous P. furiosus psiRNA 7.01 (first psiRNA encoded in CRISPR locus 7, which Northern analysis indicates is present in the native complexes, see FIG. 5). The 7.01 target RNA was cleaved at two sites (site 1 indicated with green vertical line and site 2 indicated with blue vertical line, FIG. 7B, panel 1) yielding 5′ end-labeled products of 27 and 21 nucleotides (indicated with corresponding green and blue arrowheads, FIG. 7A, panel 1). Cleavage activity was lost in the presence of 0.1 mM EDTA indicating that the enzyme depends on divalent cations, and was restored by the addition of 1 mM Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺, Ni²⁺ or Fe²⁺ with no detectable change in cleavage sites with any of the metals, but was not supported by Co²⁺ or Cu²⁺. Cleavage did not require sequences extending beyond the 37-nucleotide region of complementarity and occurred at the same two sites in the target RNA lacking sequence extensions (FIG. 7, panel 5). No activity was observed toward RNAs that lacked homology with known P. furiosus psiRNAs, including the reverse 7.01 target sequence, antisense 7.01 target sequence, and a box C/D RNA (FIG. 7, panels 2, 6, and 8). A single-stranded DNA 7.01 target sequence was also not cleaved (FIG. 7, panel 3). Pre-annealing a synthetic psiRNA 7.01 to the 7.01 target RNA (to form a double-stranded RNA target) blocked cleavage by the psiRNPs in the sample (FIG. 7, panel 4). Finally, we tested a target for endogenous P. furiosus psiRNA 6.01 and observed cleavage that generates 2 products of the same sizes observed for the 7.01 target RNA (FIG. 7, panel 7).

These results reveal the presence of cleavage activity in P. furiosus that is specific for single-stranded RNAs that are complementary to psiRNAs. The activity is associated with a purified fraction that contains 2 mature psiRNA species and 7 RAMP module (Cmr) proteins.

Cleavage of the target RNA occurs a fixed distance from the 3′ end of the psiRNA. To investigate the mechanism of psiRNA-directed RNA cleavage, we analyzed the results of cleavage assays with a series of 6-nucleotide truncations of the 7.01 target RNA (FIG. 8A). We found that the target RNA truncations analyzed did not affect the locations of the two cleavage sites. The full-length 7.01 target RNA is cleaved at sites 1 and 2 to generate 14- and 20-nucleotide 5′ end-labeled products, respectively (FIGS. 3 and 4A). The 3′ end-truncated target RNAs were cleaved at the same two sites to yield the same two 5′ end-labeled cleavage products (except where truncation eliminated cleavage site 2, Δ20-37, FIG. 8A). On the other hand, in the case of the 5′ end-truncated target RNAs, cleavage at the same sites would generate shorter 5′ endlabeled cleavage products. The 14-nucleotide product that results from cleavage of the Δ1-6 target RNA at site 2 was observed (FIG. 8A), but cleavage at site 1 could not be assessed because the size of the product is below that which could be detected in the experiment. If the twelve- and eighteen-nucleotide 5′ end-truncated target RNAs were cleaved at the same two sites, the products would also be outside the range of detection, however, interestingly, very little cleavage of these RNAs was observed (FIGS. 8, Δ1-18 and Δ1-12, compare substrate band +/− complex).

Strikingly, the difference in the sizes of the two cleavage products observed with the various substrates is the same as the difference in the sizes of the two endogenous psiRNA species (6 nucleotides in both cases, FIG. 7). This size difference as well as the specific product sizes suggest that the two cleavages occur a fixed distance (14 nucleotides) from the 3′ ends of the two psiRNAs. FIG. 8B illustrates the proposed mechanism by which the 45- and 39-nucleotide psiRNAs guide cleavage at target sites 1 and 2, respectively, for each of the target RNAs analyzed here. For example, using the full-length 7.01 target RNA we observed 20- and 14-nucleotide cleavage products (FIG. 7, panel 5) suggesting cleavage of the bound target RNA 14 nucleotides from the 3′ end of the 39- and 45-nucleotide psiRNAs, respectively (FIG. 8B, F.L.). In addition, a 7-nucleotide extension at the 5′ end of the target RNA resulted in a pair of 5′ end-labeled products 27 and 21 nucleotides in length (FIG. 7, panel 1), consistent with cleavage of the substrate 14 nucleotides from the ends of the two psiRNAs (FIG. 8B, F.L.+ext). The anchor for this counting mechanism is the 3′ end of the psiRNA. While reductions in the extent of duplex formation between the 5′ end of the psiRNA and the cleavage site (3′ truncations to within 6 nucleotides of the cleavage site) did not have an observable effect on cleavage efficiency, truncations that reduced duplex formation between the 3′ end of the psiRNA and the cleavage site had a strong negative impact, suggesting that basepairing of the last 14 nucleotides of the psiRNA with the target is critical for cleavage activity. The results of these studies indicate that both of the mature psiRNA species are active in guiding target RNA cleavage by a mechanism that depends upon the distance from the 3′ end of the psiRNA.

Analysis of reconstituted Cmr-psiRNA complexes. Identification of the Cmr proteins in the purified psiRNP complex (FIG. 5) along with the evolutionary evidence for their co-function with the CRISPRs (Haft et al., 2005, PLoS Comput Biol, 1:e60; Jansen et al., 2002, Mol Microbiol, 43:1565-1575; Makarova et al., 2002, Nucleic Acids Res, 30:482-496) strongly suggests that the Cmr proteins and psiRNAs function as a complex to cleave target RNAs (FIG. 7). In order to determine whether the Cmr proteins and psiRNAs are sufficient for function (independent of other co-purifying P. furiosus components), we tested the ability of purified recombinant Cmr proteins and synthetic psiRNAs to cleave target RNAs (FIG. 9). A reconstituted set of six P. furiosus Cmr proteins (Cmr1-1, Cmr2-Cmr6) and two mature psiRNA species (45- and 39-nucleotide psiRNA 7.01) cleaved the target RNA at 2 sites generating the same size products as those observed with the isolated native complex (FIG. 9A). While both P. furiosus isoforms of the Cmr1 protein are present in the isolated complexes (FIG. 5), we found that only one of the two proteins (Cmr1-1) was required for a functional reconstituted complex (FIG. 9A), suggesting that the isoforms may perform redundant functions. No activity was observed in the absence of the psiRNAs or in the absence of the Cmr proteins (FIG. 9A), indicating that both are necessary. These results demonstrate that the RAMP module Cas proteins and psiRNAs function together to cleave complementary target RNAs. In order to determine whether all of the six Cmr proteins are essential for psiRNA guided RNA cleavage, we assayed cleavage activity in the absence of each of the individual proteins (Figure00205B). Omission of Cmr5 did not observably affect the activity of the complex (FIG. 9B). However, cleavage was significantly reduced in the absence of any one of the other proteins (FIG. 9B), indicating that 5 of the 6 RAMP module proteins are required for activity.

Finally, we had reconstituted the same cleavage activity profile observed for the native complexes using both psiRNA species (45- and 39-nucleotides) (e.g. FIG. 9A). Our model for the mechanism of cleavage predicts that each of the psiRNAs guides a distinct cleavage: the 45-nucleotide psiRNA at site 1, and the 39-nucleotide psiRNA at site 2 (see FIG. 8B). To determine whether both psiRNAs are required for activity, and whether each guides the distinct cleavage that is predicted by the model, we tested the activity of complexes reconstituted with a single psiRNA. As predicted, we found that the 45-nucleotide psiRNA guided cleavage at site 1 producing a 14-nucleotide 5′ end-labeled product, and the 39-nucleotide psiRNA guided cleavage at site 2 producing a 20-nucleotide 5′ end-labeled product (FIG. 9C). Based on our truncation analysis (FIG. 8, Δ20-37), the larger product of the cleavage guided by the 39-nucleotide psiRNA could act as a substrate for cleavage guided by the 45-nucleotide psiRNA, and consistent with this, we often obtain more of the smaller cleavage product in cleavage assays using both the native complex and the reconstituted complex containing both psiRNAs (e.g. FIG. 9A). The results of these experiments demonstrate that each of the psiRNA species is competent to form functional psiRNPs and guides cleavage 14 nucleotides from its 3′ end.

Discussion

The findings presented here reveal the mechanism of action of an RNA-protein complex implicated in a novel RNA silencing pathway that functions in invader defense in prokaryotes. Previous work had shown that both invader-specific sequences within CRISPRs and Cas protein genes are important in virus and plasmid resistance in prokaryotes (Barrangou et al., 2007, Science, 315:1709-1712; Brouns et al., 2008, Science, 321:960-964; Deveau et al., 2008, Bacteriol, 190:1390-1400). The results presented here establish how small RNAs from CRISPRs and the RAMP module Cas proteins function together to destroy RNAs recognized by the CRISPR RNAs. The major findings and models established in this work are summarized in FIG. 10.

Our findings indicate that the RAMP module of the CRISPR-Cas system silences invaders by psiRNA-guided cleavage of invader RNAs (FIG. 10). Specifically, the results indicate that psiRNAs present in complexes with the Cmr proteins recognize and bind an invader RNA such as a viral mRNA (via the psiRNA guide sequence co-opted from the invader by another branch of the CRISPR-Cas system), and that the complex then cleaves the invader RNA, destroying the message and presumably blocking the viral life cycle. The psiRNA-Cmr complexes cleave complementary RNAs (FIGS. 3 and 5). Five of the six Cmr proteins are required for target RNA cleavage (FIG. 9) and the component of the complex that provides catalytic activity remains to be determined. Cmr2 contains a predicted nuclease domain (Makarova et al., 2002, Nucleic Acids Res, 30:482-496; Makarova et al., 2006, Biol Direct, 1:7), however the other four essential proteins (Cmr1, 3, 4 and 6) belong to the RAMP superfamily, members of which have been found to be ribonucleases (Beloglazova et al., 2008, J Biol Chem, 283:20361-20371; Brouns et al., 2008, Science, 321:960-964; Carte et al., 2008, Genes Dev, 22:3489-3496).

Our results also establish a simple model for the mechanism of cleavage site selection by the psiRNA-Cmr effector complex—a 14-nucleotide ruler anchored by the 3′ end of the psiRNA (FIG. 10). We found that P. furiosus psiRNAs occur in two lengths that share a 5′ psi-tag (derived from the CRISPR repeat) and contain either ˜37 or ˜31 nucleotides of guide sequence (FIGS. 1 and 2). Both psiRNA species are associated with the Cmr effector complex (FIG. 5) and each guides cleavage at a distinct site (FIG. 9C). Analysis of the cleavage products of both psiRNAs and of a series of substrate RNAs (FIGS. 3, 4 and 5) indicates that the complex cleaves based on a 14-nucleotide counting mechanism anchored by the 3′ end of the psiRNA. The results suggest that the 3′ end of the psiRNA places the bound target RNA relative to the enzyme active site (FIG. 10). Interestingly, Argonaute 2 (a.k.a. Slicer), an enzyme with an analogous function in the eukaryotic RNAi pathway, also employs a ruler mechanism; however, in that case the site of cleavage is located ˜10-11 nucleotides from the 5′ end of the siRNA (Elbashir et al., 2001, Nature, 411:494-498; Elbashir et al., 2001, Embo J, 20:6877-6888).

FIG. 10 also illustrates the Cmr-psiRNA effector complex model that arises from the findings presented here. Both size classes of psiRNAs and all seven Cmr proteins are found in complexes in active, purified fractions (FIG. 5), however accurate RNA guided cleavage activity can be reconstituted with either psiRNA species and with a single Cmr1 isoform (FIG. 9). We hypothesize that each psiRNA associates with a single set of six Cmr proteins, and that Cmr1-1 and Cmr1-2 function redundantly in P. furiosus. Five unrelated proteins that co-purified with the complexes (Supplemental Table 1) are not essential for reconstitution of cleavage activity in vitro (FIG. 9) and are not included in our model, but could play a role in function in vivo. Recognition of the psiRNAs by the Cmr proteins, and psiRNA-Cmr complex assembly likely depend upon conserved features of the RNAs that could include 5′ and 3′ end groups and folded structure as well as the psi-tag. Our data reveal that the psiRNA-Cmr complex can utilize psiRNAs of different sizes to cleave a target RNA at distinct sites (FIG. 9C). Thus, the two size forms of psiRNAs present in P. furiosus may provide more certain and efficient target destruction.

Our data indicate that the function of the Cmr module of Cas proteins is psiRNA-guided destruction of invading target RNA. The widespread occurrence of the cmr genes in diverse archaea and bacteria indicates that invader RNA cleavage is a mechanism utilized by many prokaryotes for viral defense (Haft et al., 2005, PLoS Comput Biol, 1:e60; Jansen et al., 2002, Mol Microbiol, 43:1565-1575; Makarova et al., 2006, Biol Direct, 1:7). However, not all prokaryotes with the CRISPR-Cas system possess the Cmr module. In these numerous other organisms, it is possible that a different set of Cas proteins mediates psiRNA-guided RNA cleavage or that Cas proteins effect invader resistance by another mechanism. Indeed, very recent work indicates that the CRISPR-Cas system targets invader DNA in a strain of Staphylococcus epidermis and perhaps E. coli (Browns et al., 2008, Science, 321:960-964; Lillestol et al., 2006, Archaea, 2:59-72), which possess the Mycobacterium tuberculosis (Csm) and E. coli (Cse) subtype Cas protein modules, respectively (Haft et al., 2005, PLoS Comput Biol, 1:e60; Jansen et al., 2002, Mol Microbiol, 43:1565-1575; Makarova et al., 2006, Biol Direct, 1:7). The prokaryotes include evolutionarily distant and very diverse organisms. Diversity in the core components of the eukaryotic RNAi machinery has led to a tremendous variety of observed RNA-mediated gene silencing pathways that can act at post-transcriptional or transcriptional levels (Chapman and Carrington, 2007, Nat Rev Genet, 8:884-896; Farazi et al., 2008, Development, 135:1201-1214; Hutvagner and Simard, 2008, Nat Rev Mol Cell Biol, 9:22-32; Zaratiegui et al., 2007, Cell, 128:763-776). The diversity of Cas proteins found in CRISPR-containing prokaryotes may reflect significantly different mechanisms of CRISPR element integration, CRISPR RNA biogenesis, and invader silencing.

Example 3 Cleavage of Target Polynucleotides by a Cmr Complex Requires a psiRNA-Tag

A psiRNA with the wildtype tag (AUUGAAAG, wt) efficiently guides cleavage of a complementary RNA in the presence of Cmr1-6 (+proteins). The same psiRNA that is lacking the tag sequence (-tag) is unable to guide cleavage of the target. Mutating the tag sequence to its complement, UAACUUUC (comp. tag), also inactivates the complex. This indicates that the tag sequence, AUUGAAAG, is required for cleavage of a complementary RNA by the Cmr proteins.

Recombinant Cmr1-6 (500 μM each protein) was added to a 20 reaction using conditions described in Example 1. Proteins and psiRNAs (0.25 pmol psiRNA per reaction) were incubated for 30 minutes at 70° C. [³²P]-radiolabeled target RNAs (0.05 pmol per reaction) were added and the reactions were incubated for 1 hour and 70° C. The resulting reaction was subject to proteinase K digest by 1 μg of proteinase K at 37° C. for 15 minutes, followed by PCI extraction and Ethanol precipitation. The resulting RNAs were separated on 10×10.6 cm 15% 7M urea gels and subject to autoradiography. Decade marker (Applied Biosystems) was used to determine sizes of cleavage products.

The following RNA sequences were used, Target (for all reactions), CTGUUGTGCTCTCUGCCGCUUGGUCCGCUTUCTUCUU (SEQ ID NO:256); wt psiRNA,

AUUGAAAGUUGUAGUAUGCGGUCCUUGCGGCUGAGAGCACUUCAG (SEQ ID NO: 204);

-tag psiRNA, UUGUAGUAUGCGGUCCUUGCGGCUGAGAGCACUUCAG (SEQ ID NO:205); complement tag psiRNA,

UAACUUUCUUGUAGUAUGCGGUCCUUGCGGCUGAGAGCACUUCAG (SEQ ID NO:206). Example 4 The psi-tag Allows for Rational Design of psiRNAs that Guide Inactivation of Novel Targets

psiRNAs were designed to target two novel RNA sequences (not targeted by known naturally occurring psiRNAs): “exogenous target”, which does not correspond to any known naturally occurring sequence, and the bla target, which corresponds to the first 37 nucleotides of the β-lactamase or bla mRNA, which encodes a protein responsible for common forms of antibiotic resistance in bacteria. The psiRNAs were constructed by addition of the P. furiosus psi-tag sequence, AUUGAAAG, to 37 nucleotides of guide sequence that is complementary to the targeted sequence. The results are shown in the figure below. The psiRNAs and the Cmr proteins were incubated with the target RNAs. The psiRNA directed against the exogenous target (“exogenous psiRNA”) directed cleavage of the exogenous target, but not the bla target RNA. The psiRNA directed against the bla target (“bla psiRNA”) directed cleavage of the bla target, but not the exogneous target RNA. These results indicate that psiRNAs can be rationally designed to direct cleavage of novel target RNAs by the Cmr complex.

Cmr complex assays were performed as described in Example 1. Cmr1-6 (500 μM) was incubated with 0.25 pmol of psiRNA (described below) for 30 minutes at 70° C. in conditions described in Example 1. Radiolabeled target RNA (0.05 pmol, sequences below) was added and the reaction was allowed to continue at 70° C. for 1 hour. One microgram of proteinase K was added and incubated at 37° C. for 15 minutes. The resulting RNAs were subject to PCI extraction and ethanol precipitation. The purified RNAs were separated on 10×10.6 cm 15% 7M urea gels. The gels were dried and subject to autoradiography. Decade marker (Applied Biosystems) was used to determine sizes of cleavage products.

The following RNA sequences were used: exogenous psiRNA, AUUGAAAGCUGAAGUGCUCUCAGCCGCAAGGACCGCAUACUACAA (SEQ ID NO:20);

exogenous target, UUGUAGUAUGCGGUCCUUGCGGCUGAGAGCACUUCAG (SEQ ID NO:21); bla psiRNA, AUUGAAAGCUGAAGUGCUCUCAGCCGCAAGGACCGCAUACUACAA (SEQ ID NO:22); and bla target,

AUGAGUAUUCAACAUUUCCGUGUCGCCCUUAUUCCCU (SEQ ID NO:23).

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and/or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. 

1-56. (canceled)
 57. An isolated polynucleotide comprising a 5′ region and a 3′ region, wherein the 5′ region comprises a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer present in a microbe comprising a CRISPR locus, and wherein the 3′ region comprises at least 18 nucleotide.
 58. A vector encoding the isolated polynucleotide of claim
 57. 59. The isolated polynucleotide of claim 57 wherein the isolated polynucleotide is RNA.
 60. The isolated polynucleotide of claim 57 wherein the psiRNA-tag has at least 80% sequence similarity with 5′-ATTGAAAS, wherein S is G or C.
 61. The isolated polynucleotide of claim 57 wherein the 3′ region comprises at least 31 nucleotides or at least 37 nucleotides.
 62. A method for inactivating a target polynucleotide in a microbe comprising: introducing into the microbe a psiRNA comprising a 5′ region and a 3′ region, wherein the 5′ region is a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer, wherein the 3′ region comprises between 18 and 75 nucleotides, and wherein the 3′ region is substantially complementary to a portion of the target polynucleotide, wherein the target polynucleotide is inactivated.
 63. The method of claim 62 wherein the psiRNA is introduced into the cell as an RNA polynucleotide.
 64. The method of claim 62 wherein the psiRNA is introduced into the cell as a DNA polynucleotide encoding the psiRNA.
 65. The method of claim 62 wherein the psiRNA-tag has at least 80% sequence similarity with 5′-AUUGAAAS, wherein S is G or C.
 66. The method of claim 62 wherein the target polynucleotide is an endogenous polynucleotide.
 67. The method of claim 62 wherein the target polynucleotide is an exogenous polynucleotide.
 68. The method of claim 67 wherein the exogenous polynucleotide is a bacteriophage polynucleotide.
 69. The method of claim 62 wherein the psiRNA-tag nucleotide sequence is chosen from a CRISPR locus present in the microbe.
 70. A method for cleaving a target polynucleotide comprising: incubating under suitable conditions a composition comprising: a target polynucleotide; a psiRNA comprising a 5′ region and a 3′ region, wherein the 5′ region is a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer, wherein the 3′ region comprises 5′-NNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNN (SEQ ID NO:24) or 5′-NNNNNNNNNNNNNNNNNNNNNNN↓NNNNNNNNNNNNNNNNNNNN(SEQ ID NO:25), wherein the 3′ region is substantially complementary to a portion of the target polynucleotide; and a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, and a Cmr6 polypeptide; wherein the target polynucleotide is cleaved in the region that is substantially complementary to the region of SEQ ID NO:24 or SEQ ID NO:25.
 71. The method of claim 70 wherein the psiRNA-tag has at least 80% sequence similarity with 5′-AUUGAAAS, wherein S is G or C.
 72. The method of claim 70 wherein the incubating is at a temperature greater than 30° C.
 73. The method of claim 70 wherein the amino acid sequence of Cmr1 and the amino acid sequence of SEQ ID NO:4 have at least 80% identity, wherein the amino acid sequence of Cmr2 and the amino acid sequence of SEQ ID NO:8 have at least 80% identity, wherein the amino acid sequence of Cmr3 and the amino acid sequence of SEQ ID NO:10 have at least 80% identity, wherein the amino acid sequence of Cmr4 and the amino acid sequence of SEQ ID NO:12 have at least 80% identity, wherein the amino acid sequence of Cmr6 and the amino acid sequence of SEQ ID NO:16 have at least 80% identity, and wherein the polypeptides have endonuclease activity.
 74. The method of claim 70 wherein the method is in vitro.
 75. The method of claim 70 wherein the method is in vivo.
 76. The method of claim 70 wherein the method occurs within a Pyrococcus furiosus, a Sulfolobus solfataricus, or a S. tokodaii cell. 